Uses of adenosine base editors

ABSTRACT

The disclosure provides methods and compositions for treating blood diseases/disorders, such as sickle cell disease, hemochromatosis, hemophilia, and beta-thalassemia. For example the disclosure provides therapeutic guide RNAs that target the promotor of HBG1/2 to generate point mutations that increase expression of fetal hemoglobin. As another example, the disclosure provides therapeutic guide RNAs that target mutations in HBB, Factor VIII, and HFE to treat sickle cell disease, beta-thalassemia, hemophilia and hemochromatosis. The disclosure also provides fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA. In some embodiments, the fusion proteins are in complex with nucleic acids, such as guide RNAs (gRNAs), which target the fusion proteins to a DNA sequence (e.g., an HBG1 or HBG2 protmoter sequence, or an HFE, GBB, or F8 gene sequence). Such complexes may be useful for increasing expression of fetal hemoglobin or correcting a poing mutation (e.g., C282Y) in HFE.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of international PCT application, PCT/US2018/056146, filed Oct. 16, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S. Ser. No. 62/573,127, filed on Oct. 16, 2017, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. EB022376, HG009490, and GM118062 awarded by the National Institutes of Health, and Grant No. HR0011-17-2-0049 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases. Since many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, an A to G or a T to C change in a specific codon of a gene associated with a disease), the development of a programmable way to achieve such precise gene editing represents both a powerful new research tool, as well as a potential new approach to gene editing-based therapeutics.

SUMMARY OF THE INVENTION

The disclosure provides methods and compositions for treating blood diseases/disorders, such as sickle cell disease, hemochromatosis, hemophilia, and beta-thalassemia. For example, the disclosure provides therapeutic guide RNAs that target the promotor of HBG1/2 to generate point mutations that increase expression of fetal hemoglobin. As another example, the disclosure provides therapeutic guide RNAs that target mutations (e.g., pathogenic mutaions) in HBB, Factor VIII, and HFE to treat sickle cell disease, beta-thalassemia (e.g., Hb C and Hb E), hemophilia and hemochromatosis. The guide RNAs provided herein can be complexed with a base editor protein (e.g., an adenosine base editor) to generate a point mutation in a gene or gene promoter, which can correct a pathogenic mutation, generate a non-pathogenic point mutation, or modulate (e.g., increase) expression of a gene.

Provided herein are compositions, kits, and methods of modifying a polynucleotide (e.g., DNA) using an adenosine deaminase and a nucleic acid programmable DNA binding protein (e.g., Cas9). Some aspects of the disclosure provide nucleobase editing proteins which catalyze hydrolytic deamination of adenosine (forming inosine, which base pairs like guanine (G)) in the context of DNA. There are no known naturally occurring adenosine deaminases that act on DNA. Instead, known adenosine deaminases act on RNA (e.g., tRNA or mRNA). To overcome this drawback, the first deoxyadenosine deaminases were evolved to accept DNA substrates and deaminate deoxyadenosine (dA) to deoxyinosine. Such adenosine deaminases are described in International Application No.: PCT/US2017/045,381, filed Aug. 3, 2017; the entire contents of which are hereby incorporated by reference. The adenosine deaminase acting on tRNA (ADAT) from Escherichia coli (TadA, for tRNA adenosine deaminase A), was covalently fused to a dCas9 or a Cas9 nickase domain, and fusion proteins containing mutations in the deaminase portion of the construct were assembled. In addition to E. coli TadA (ecTadA), other naturally occurring adenosine deaminases, such as human ADAR (adenosine deaminase acting on RNA), mouse ADA (adenosine deaminase), and human ADAT2, may be fused to a dCas9 or Cas9 nickase domain to generate adenosine nucleobase editor (ABE) fusion protein constructs. The directed evolution of these fusion proteins resulted in programmable adenosine base editors that efficiently convert target A-T base pairs to G-C base pairs with low off-target modifications and a low rate of indel (stochastic insertion or deletion) formation, especially when compared to current Cas9 nuclease-mediated HDR methods of genome editing. The ABEs disclosed herein can be used to both correct disease-associated point mutations and to introduce disease-suppressing point mutations (e.g., single nucleotide polymorphisms).

Mutations in the deaminase domain of nucleobase editing proteins were made by evolving adenosine deaminases. For example, ecTadA variants that are capable of deaminating adenosine in DNA include one or more of the following mutations: W23L, W23R, R26G, H36L, N37S, P48S, P48T, P48A, I49V, R51L, N72D, L84F, S97C, D108N, A106V, H123Y, G125A, A142N, S146C, D147Y, R152H, R152P, E155V, I156F, K157N, and K161T of SEQ ID NO: 1. It should be appreciated however, that homologous mutations may be made in other adenosine deaminases to generate variants that are capable of deaminating adenosine in DNA. FIG. 7 illustrates ecTadA variants that may be useful for the methods disclosed herein.

In the examples provided herein, exemplary nucleobase editors having the general structure of an evolved fusion protein, such as ecTadA(D108X; X=G, V, or N)-XTEN-nCas9, catalyzed A to G transition mutations in cells such as eukaryotic cells (e.g., Hek293T mammalian cells). In other examples exemplary nucleobase editors contain two ecTadA domains and a nucleic acid programmable DNA binding protein (napDNAbp). The two ecTadA domains may be the same (e.g., a homodimer), or two different ecTadA domains (e.g., a heterodimer (e.g., wild-type ecTadA and ecTadA(A106V/D108N))). For example nucleobase editors may have the general structure ecTadA-ecTadA*-nCas9, where ecTadA* represents an evolved ecTadA comprising one or more mutations of SEQ ID NO:1. Additional examples of nucleobase editors containing ecTadA variants provided herein demonstrate an improvement in performance of the nucleobase editors in mammalian cells.

Without wishing to be bound by any particular theory, the adenosine nucleobase editors described herein work by using ecTadA variants to deaminate A bases in DNA, causing A to G mutations via inosine formation. Inosine preferentially hydrogen bonds with C, resulting in A to G mutation during DNA replication. When covalently tethered to Cas9 (or another nucleic acid programmable DNA binding protein), the adenosine deaminase (e.g., ecTadA) is localized to a gene of interest and catalyzes A to G mutations in the ssDNA substrate. This editor can be used to target and revert single nucleotide polymorphisms (SNPs) in disease-relevant genes, which require A to G reversion. This editor can also be used to target and revert single nucleotide polymorphisms (SNPs) in disease-relevant genes, which require T to C reversion by mutating the A, opposite of the T, to a G. The T may then be replaced with a C, for example by base excision repair mechanisms, or may be changed in subsequent rounds of DNA replication. Thus, the adenosine base editors described herein may deaminate the A nucleobase to give a nucleotide sequence that is not associated with a disease or disorder. In some aspects, the adenosine base editors described herein may be useful for deaminating an adenosine (A) nucleobase in a gene promoter. In some embodiments, deamination leads to induce transcription of the gene. The induction of transcription of a gene leads to an increase in expression of the protein encoded by the gene (e.g., the gene product). A guide RNA (gRNA) bound to the base editor comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence in the promoter of the HBG1 and/or the HBG2 gene. In some embodiments, the target nucleic acid sequence in the promotor comprises the nucleic acid sequence 5′-CTTGGGGGCCCCTTCCCCACACTA-3′ (SEQ ID NO: 838), 5′-CTTGGGGGCCCCTTCCCCACACT-3′ (SEQ ID NO: 839), 5′-CTTGGGGGCCCCTTCCCCACAC-3′ (SEQ ID NO: 840), 5′-CTTGGGGGCCCCTTCCCCACA-3′ (SEQ ID NO: 841), 5′-CTTGGGGGCCCCTTCCCCAC-3′ (SEQ ID NO: 842), 5′-CTTGGGGGCCCCTTCCCCA-3′ (SEQ ID NO: 843), 5′-CTTGGGGGCCCCTTCCCC-3′ (SEQ ID NO: 844), or 5′-CTTGGGGGCCCCTTCCC-3′ (SEQ ID NO: 845). In some embodiments, the target nucleic acid sequence in the promoter comprises the nucleic acid sequence 5′-CTTGGGGGCCCCTTCCCCAC-3′ (SEQ ID NO: 842). In some embodiments, the target nucleic acid sequence in the promoter comprises a T at nucleic acid position 14 (shown in bold) of any one of SEQ ID NOs: 838-845, and the deamination of the A nucleobase that is complementary to the T at position 14 results in a T to C mutation in the target nucleic acid sequence. In some embodiments, the target nucleic acid further comprises 5′-CCT-3′ at the 5′ end of any one of SEQ ID NOs: 838-845. In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 26 contiguous nucleic acids that are 100% complementary to the target nucleic acid sequence in the promoter. In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence 5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 846), 5′-CAUGUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 847), 5′-AUGUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 848), 5′-UGUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 849), 5′-GUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 850), 5′-UGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 851) 5′-GGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 852), or 5′-GGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 853). In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence 5′-GUGGGGAAGGGGCCCCCAAG-3′ (SEQ ID NO: 850). In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 254-280. In some embodiments, the base editor comprises a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) an adenosine deaminase is used in combination with a guide RNA that is complementary to a target sequence of interest to demainate a nucleobase. In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase (nCas9). The adenosine deaminse may be any adenosine deaminase described herein. In some embodiments, the adenosine demainse is an E. coli TadA (ecTadA) comprising one or more mutations of SEQ ID NO: 1. In some embodiments, the adenosine deaminse is an ecTadA comprising the amino acid sequence of SEQ ID NO: 1.

Thus, in some aspects, the base editor and guide RNA complexes described herein may be useful for treating a disease or a disorder caused by a C to T mutation in the promoter of the HBG1 and/or the HBG2 gene. In some embodiments, the disease or disorder is one of the blood. In certain embodiments, the disease or disorder is anemia. In some embodiments, the anemia is sickle-cell anemia. For example, a −198 C to T mutation in the promoter of the HBG1 and/or the HBG2 gene leads to a decrease in γ-globin expression levels, which can promote the development of sickle cell disease (SCD) and β-thalassemia. The deamination of the adenosine nucleobase in the promoter results in a T-A base pair in the promoter being mutated to a C-G base pair in the promoter of the HBG1 and/or the HBG2 gene. The deamination of the adenosine nucleobase promotes a sequence that is promoted with hereditary persistence to fetal hemoglobin (HPFH). In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in transcription of the HBG1 gene. In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in the amount of the HBG1 protein. In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in transcription of the HBG2 gene.

In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in the amount of the HBG2 protein. In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in transcription of both the HBG1 and HBG2 genes. In some embodiments, deaminating the adenosine nucleobase in the promoter leads to an increase in the amount of both the HBG1 and HBG2 proteins.

In yet another aspect, the adenosine base editors described herein may be useful for deaminating an adenosine (A) nucleobase in a gene to correct a point mutation in the gene. In some embodiments, the gene may comprise a mutation in a codon that results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. A guide RNA (gRNA) bound to the base editor comprises a guide sequence that is complementary to a target nucleic acid sequence in the gene. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence in the HFE gene. In some embodiments, the HFE gene comprises a C to T mutation. Deamination of the A nucleobase complementary to the T by a base editor and guide RNA complex described herein corrects the C to T mutation (for example, see FIG. 7 ). In some embodiments, the target nucleic acid sequence in the promotor comprises the nucleic acid sequence. In some embodiments, the HFE gene encodes a human hemochromatosis (HFE) protein comprising a Cys to Tyr mutation (e.g., C282Y). Deamination of the A nucleobase complementary to the T by a base editor and guide RNA complex described herein corrects the Cys to Tyr mutation in the HFE protein, resulting in expression of the wild-type protein. In some embodiments, the target nucleic acid sequence in the HFE gene comprises the nucleic acid sequence 5′-GGGTGCTCCACCTGGTACGTATAT-3′ (SEQ ID NO: 854), 5′-GGGTGCTCCACCTGGTACGTATA-3′ (SEQ ID NO: 855), 5′-GGGTGCTCCACCTGGTACGTAT-3′ (SEQ ID NO: 856), 5′-GGGTGCTCCACCTGGTACGTA-3′ (SEQ ID NO: 857), 5′-GGGTGCTCCACCTGGTACGT-3′ (SEQ ID NO: 858), 5′-GGGTGCTCCACCTGGTACG-3′ (SEQ ID NO: 859), 5′-GGGTGCTCCACCTGGTAC-3′ (SEQ ID NO: 860), or 5′-GGGTGCTCCACCTGGTA-3′ (SEQ ID NO: 861). In some embodiments, the target nucleic acid further comprises 5′-CCT-3′ at the 5′ end of any one of the nucleic acid sequences of SEQ ID NOs: 854-861. In some embodiments, the target nucleic acid sequence in the HFE gene comprises the nucleic acid sequence 5′-GGGTGCTCCACCTGGTACGT-3′ (SEQ ID NO: 858). In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 26 contiguous nucleic acids that are 100% complementary to the target nucleic acid sequence in the HFE gene. In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence 5′-AUAUACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 862), 5′-UAUACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 863), 5′-AUACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 864), 5′-UACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 865), 5′-ACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 866), 5′-CGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 867), 5′-GUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 868), or 5′-UACCAGGUGGAGCACCC-3′ (SEQ ID NO: 869). In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of any one of the nucleic acid sequences of SEQ ID NOs: 862-869. In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence 5′-GACGUACCAGGUGGAGCACCC-3′ (SEQ ID NO: 870). In some embodiments, the base editor comprises a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) an adenosine deaminase is used in combination with a guide RNA that is complementary to a target sequence of interest to demainate a nucleobase. In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase (nCas9). The adenosine deaminse may be any adenosine deaminase described herein. In some embodiments, the adenosine demainse is an E. coli TadA (ecTadA) comprising one or more mutations of SEQ ID NO: 1. In some embodiments, the adenosine deaminse is an ecTadA comprising the amino acid sequence of SEQ ID NO: 1.

Thus, in some aspects, the base editor and guide RNA complexes described herein may be useful for treating a disease or a disorder caused by a C to T mutation in the HFE gene. In some embodiments, the disorder is an iron storage disorder. In some embodiments, the iron storage disorder is hereditary hemochromatosis (HHC). In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in a T-A base pair in the HFE gene being mutated to a C-G base pair in the HFE gene. In some embodiments, deaminating the adenosine nucleobase in the HFE gene leads to an increase in an HFE protein function transcribed from the HFE gene. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in correcting a sequence associated with hereditary hemochromatosis (HHC). In some embodiments, deaminating the adenosine nucleobase in the HFE gene ameliorates one or more symptoms of the iron storage disorder.

In some embodiments, the adenosine deaminases provided herein are capable of deaminating an adenosine in a nucleic acid molecule. In some embodiments, the method of deaminating an adenosine nucleobase comprises contacting the nucleic acid molecule in a cell with a complex comprising (i) an adenosine base editor and (ii) a guide RNA in vitro. In some embodiments, the method of deaminating an adenosine nucleobase comprises contacting the nucleic acid molecule in cell with a complex comprising (i) an adenosine base editor and (ii) a guide RNA in vivo. In some embodiments, the method of deaminating an adenosine nucleobase comprises contacting the nucleic acid molecule in a cell with a complex comprising (i) an adenosine base editor and (ii) a guide RNA, wherein the cell is in a subject. In some embodiments, the cell is an immortalized lymphoblastoid cell (LCL). Other aspects of the disclosure provide fusion proteins comprising a Cas9 domain and an adenosine deaminase domain, for example, an engineered deaminase domain capable of deaminating an adenosine in DNA. In some embodiments, the fusion protein comprises one or more of a nuclear localization sequence (NLS), an inhibitor of inosine base excision repair (e.g., dISN), and/or one or more linkers.

In some aspects, the disclosure provides an adenosine deaminase capable of deaminating an adenosine in a deoxyribonucleic acid (DNA) substrate. In some embodiments, the adenosine deaminase is from a bacterium, for example, E. coli or S. aureus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the adenosine deaminase comprises a D108X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, X is G, N, V, A, or Y. In some embodiments, the adenosine deaminase comprises a D108N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, X is V, G, I, L, or A. In some embodiments, the adenosine deaminase comprises a A106V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106X and a D108X mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, the adenosine deaminase comprises a A106V and a D108N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminse comprises a A106X, D108X, D147X, E155X, L84X, H123X, and I156X mutation, or corresponding mutations in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, the adenosine deaminse comprises a A106V, D108N, D147Y, E155V, L84F, H123Y, and I156F mutation, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106X, D108X, D147X, E155X, L84X, H123X, I156X, H36X, R51X, S146X, and K157X mutation, or corresponding mutations in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, the adenosine deaminse comprises a A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, and K157N mutation, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine comprises a A106X, D108X, D147X, E155V, L84X, H123X, I156X, H36X, R51X, S146X, K157X, W23X, P48X, and R152X mutation, or the corresponding mutations in another adenosine deaminase, wherein X is any amino acid other than the amino acid found in the wild-type protein. In some embodiments, the adenosine deaminase comprises a A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, W23R, P48A, and R152P mutation, or the corresponding mutations in another adenosine deaminase. It should be appreciated that the adenosine deaminases provided herein may contain one or more of the mutations provided herein in any combination.

In another aspect, the disclosure provides adenosine nucleobase editors (ABEs) with broadened target sequence compatibility. In general, native ecTadA deaminates the adenine in the sequence UAC (e.g., the target sequence) of the anticodon loop of tRNA^(Arg). Without wishing to be bound by any particular theory, in order to expand the utility of ABEs comprising one or more ecTadA domains, such as any of the adenosine deaminases provided herein, the adenosine deaminase proteins were optimized to recognize a wide variety of target sequences within the protospacer sequence without compromising the editing efficiency of the adenosine nucleobase editor complex. In some embodiments, the target sequence is 5′-NAN-3′, wherein N is T, C, G, or A. For example, target sequences are shown in FIG. 3A. In some embodiments, the target sequence comprises 5′-TAC-3′. In some embodiments, the target sequence comprises 5′-GAA-3′.

In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 707. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 708. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 709. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 710. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 711.

In another aspect, the present disclosure provides pharmaceutical compositions comprising a complex comprising a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), (ii) an adenosine deaminase, and (iii) a guide RNA that directs the fusion protein to a target sequence of interest; and optionally a pharmaceutically acceptable excipient. The adenosine deaminase can be any of the adenosine deaminase domains described herein, or any combination thereof. In some embodiments, the napDNAbp is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase (nCas9). In some embodiments, the guide RNA comprises a guide nucleic acid sequence that directs the fusion protein to the promoter of the HBG1 and/or the HBG2 gene. In some embodiments, the guide nucleic acid sequence comprises a nucleic acid sequence of SEQ ID NOs: 846-853. In some embodiments, the guide RNA comprises a nucleic acid sequence that directs the fusion protein to the HFE gene. In some embodiments, the guide nucleic acid sequence comprises a nucleic acid sequence of SEQ ID NOs: 862-870. In some embodiments, the pharmaceutical composition is administered to a subject in need thereof (e.g., to treat a disease or disorder). In some embodiments, the subject has a blood disease. In some embodiments, the blood disease is anemia. In some embodiments, the anemia is sickle cell anemia. In some embodiments, the subject has an iron storage disorder. In some embodiments, the iron storage disorder is hereditary hemochromatosis (HHC). In yet another aspect, the present disclosure provides kits comprising a nucleic acid construct, comprising (a) a nucleic acid sequence encoding a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) an adenosine deaminase; (b) a heterologous promoter that drives expression of the sequence of (a); and (c) an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone. The adenosine deaminase can be any of the adenosine deaminase domains described herein, or any combination thereof. In some embodiments, the napDNAbp is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase (nCas9). In some embodiments, the guide RNA comprises a nucleic acid sequence that directs the fusion protein to the promoter of the HBG1 and/or the HBG2 gene. In some embodiments, the target nucleic acid sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 838-845. In some embodiments, the guide RNA comprises a nucleic acid sequence that directs the fusion protein to the HFE gene. In some embodiments, the target nucleic acid sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 854-861.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C show scope and overview of base editing by an A•T to G•C base editor (ABE). FIG. 1A: Relative distribution of the base pair changes required to correct known pathogenic human SNPs in the ClinVar database. FIG. 1B: The hydrolytic deamination of adenosine (A) forms inosine (I), which is read as guanosine (G) by polymerase enzymes. FIG. 1C: ABE-mediated A•T to G•C base editing strategy. ABEs contain a hypothetical deoxyadenosine deaminase, which is not known to exist in nature, and a catalytically impaired Cas9. They bind a double-stranded DNA target of interest in a guide RNA-programmed manner, exposing a small bubble of single stranded DNA in the “R-loop” complex. The deoxyadenosine deaminase domain catalyzes deoxyadenosine to deoxyinosine formation within this single-stranded bubble. Following DNA repair or replication, the original A•T base pair is replaced with a G•C base pair at the target site.

FIGS. 2A to 2C show protein evolution and engineering of ABEs. FIG. 2A: Strategy to evolve a DNA deoxyadenosine deaminase starting from an RNA adenosine deaminase. A library of E. coli cells each harbor a plasmid expressing a mutant ecTadA (TadA*) gene fused to catalytically dead Cas9 (dCas9) and a selection plasmid expressing a defective antibiotic resistance gene requiring one or more A•T to G•C mutations to restore antibiotic resistance. Mutations from TadA* variants surviving each round of mutation and selection were imported into a mammalian ABE architecture and assayed in human cells for programmable A•T to G•C base editing activity. FIG. 2B: Genotypes of a subset of evolved ABEs arising from each round of evolution. For a list of all 57 evolved ABE genotypes characterized in this work, see FIG. 7 . The dimerization state (monomer, homodimer of evolved TadA* domains, or heterodimer of wild-type TadA—evolved TadA*) and linker length (in number of amino acids) are also listed. Mutations are colored based on the round of evolution from which they were identified. FIG. 2C: Three views of the E. coli TadA deaminase structure (PDB 1Z3A) aligned with the structure of S. aureus TadA (not shown) complexed with tRNAArg2 (PDB 2B3J). The UAC anticodon loop of the tRNA is the endogenous substrate of wild-type TadA. The TadA dimer and the tRNA anticodon loop are shown on the left; residues near the 2′ hydroxyl group of the ribose of the U upstream of the substrate adenosine are shown in the middle; residues that mutated during ABE evolution and surround the UAC substrate are shown on the right. Wildtype residues are colored to correspond to the mutations emerging from each round of evolution, and to the genotype table in FIG. 2B.

FIGS. 3A to 3C show early- and mid-stage evolved ABEs mediate A•T to G•C base editing at human genomic DNA sites. FIG. 3A: Table of 19 human genomic DNA test sites (left) with corresponding locations on human chromosomes (right). The sequence context (target motif) of the edited A in red is shown for each site. PAM sequences are shown in blue.

FIG. 3B: A•T to G•C base editing efficiencies in HEK293T cells of round 1 and round 2 ABEs at six human genomic DNA sites. FIG. 3C: A•T to G•C base editing efficiencies in HEK293T cells of round 3, round 4, and round 5 ABEs at six human genomic DNA sites. ABE2.9 editing is shown for comparison. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. Homodimer indicates fused TadA*-TadA*-Cas9 nickase architecture; heterodimer indicates fused wtTadA-TadA*-Cas9 nickase architecture. Sequences correspond from top to bottom to SEQ ID NOs: 91-109.

FIGS. 4A and 4B show late-stage evolved ABEs mediate genomic DNA editing with greater activity and broader sequence compatibility. FIG. 4A: A•T to G•C base editing efficiencies in HEK293T cells of round 6 and round 7 ABEs at six human genomic DNA sites. ABE5.3 editing is shown for comparison. FIG. 4B: A•T to G•C base editing efficiencies in HEK293T cells of round 6 and round 7 ABEs at an expanded set of human genomic sites. Sites 1-17 collectively include every possible NAN sequence context flanking the target A. ABE5.3 editing is shown for comparison. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. All ABEs shown in FIGS. 4A and 4B are heterodimers of the wtTadA-TadA*-Cas9 nickase architecture.

FIGS. 5A to 5C show activity window and product purity of late-stage ABEs. FIG. 5A: Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1-9 in two human genomic DNA sites that together place an A at each of these positions. Values are normalized to the maximum observed efficiency at each of the two sites for each ABE=1. FIG. 5B: Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1-18 and 20 across all 19 human genomic DNA sites tested. Values are normalized to the maximum observed efficiency at each of the 19 sites for each ABE=1. FIG. 5C: Product distributions at two representative human genomic DNA sites in HEK293T cells treated with ABE7.10 or ABE7.9 and the corresponding sgRNA, or in untreated HEK293T cells. Indel frequencies are shown at the right. For FIGS. 5A and 5B, values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. Sequences correspond from top to bottom to SEQ ID NOs: 110-111.

FIGS. 6A to 6C show comparison of ABE7.10-mediated base editing and Cas9-mediated HDR, and application of ABE7.10 to two disease-relevant SNPs. FIG. 6A: A•T to G•C base editing efficiencies in HEK293T cells treated either with ABE7.10, or with Cas9 nuclease and an ssDNA donor template (following the CORRECT HDR method 41) targeted to five human genomic DNA sites. FIG. 6B: Comparison of indel formation in HEK293T cells treated as described in FIG. 6A. FIG. 6C: Application of ABE to install a disease-suppressing SNP, or to correct a disease inducing SNP. Top: ABE7.10-mediated-198TaC mutation (on the strand complementary to the one shown in the sequencing data tables) in the promoter region of HBG1 and HBG2 genes in HEK293T cells. The target A is at position 7 of the protospacer. Bottom: ABE7.10-mediated reversion of the C282Y mutation in the HFE gene in LCL cells. This mutation is a common cause of hereditary hemochromatosis. The target A is at position 5 of the protospacer.

FIG. 7 shows genotype table of all ABEs described in this work. Mutations are colored based on the round of evolution in which they were identified.

FIGS. 8A to 8D show base editing efficiencies of additional early-stage ABE variants. FIG. 8A: A•T to G•C base editing efficiencies in HEK293T cells of various wild-type RNA adenine deaminases fused to Cas9 nickase at six human genomic target DNA sites. Values reflect the mean and standard deviation of three biological replicates performed on different days. FIG. 8B: A•T to G•C base editing efficiencies in HEK293T cells of ABE2 editors with altered fusion orientations and linker lengths at six human genomic target DNA sites. FIG. 8C: A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 editors fused to catalytically inactivated alkyl-adenosine glycosylase (AAG) or endonuclease V (EndoV), two proteins that bind inosine in DNA.

FIG. 8D: A•T to G•C base editing efficiencies of ABE2.1 in HAP1 cells at site 1 with or without AAG. Values and error bars in FIGS. 8B and 8C reflect the mean and s.d. of three independent biological replicates performed on different days.

FIG. 9 shows high-throughput DNA sequencing analysis of HEK293T cells treated with ABE2.1 and sgRNAs targeting each of six human genomic sites. One representative replicate is shown. Data from untreated HEK293T cells are shown for comparison.

FIGS. 10A to 10C show base editing efficiencies of additional ABE2 and ABE3 variants, and the effect of adding A142N to TadA*-dCas9 fusions on antibiotic selection survival in E. coli. FIG. 10A: A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 variants with different engineered dimeric states. Data plotted is the mean value of three biological replicates. FIG. 10B: A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE3.1 variants differing in their dimeric state (homodimer of TadA*-TadA*-Cas9 nickase, or heterodimer of wild-type TadA-TadA*-Cas9 nickase), in the length of the TadA-TadA linker, and in the length of the TadA-Cas9 nickase linker. See FIG. 7 for ABE genotypes and architectures. FIG. 10C: Colony-forming units on 2×YT agar with 256 μg/mL of spectinomycin of E. coli cells expressing an sgRNA targeting the I89T defect in the spectinomycin resistance gene and a TadA*-dCas9 editor lacking or containing the A142N mutation identified in evolution round 4 Successful A•T to G•C base editing at the target site restores spectinomycin resistance. Values and error bars in FIGS. 10A and 10B reflect the mean and s.d. of three independent biological replicates performed on different days.

FIGS. 11A and 11B show base editing efficiencies of additional ABE5 variants. FIG. 11A: A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of two ABE3.1 variants with two pairs of mutations isolated from spectinomycin selection of the round 5 library. FIG. 11B: A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE5 variants with different linker lengths. See FIG. 7 for ABE genotypes and architectures. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

FIGS. 12A to 12C show base editing efficiencies of ABET variants at 17 genomic sites. A•T to G•C base editing efficiencies in HEK293T cells at 17 human genomic target DNA sites of ABE7.1-7.5 (FIG. 12A), and ABE7.6-7.10 (FIG. 12B). See FIG. 7 for ABE genotypes and architectures. FIG. 12C: A•T to G•C base editing efficiencies in U2OS cells at six human genomic target DNA sites of ABE7.8-7.10. The lower editing efficiencies observed in U2OS cells compared with HEK293T cells are consistent with transfection efficiency differences between the two cell lines; typical transfection efficiencies of ˜40 to 55% were observed in U2OS cells under the conditions used in this study, compared to 65-80% in HEK293T cells. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

FIG. 13 shows rounds of evolution and engineering increased ABE processivity. The calculated mean normalized linkage disequilibrium (LD) between nearby target As at 6 to 17 human genomic target DNA sites for the most active ABEs emerging from each round of evolution and engineering. Higher LD values indicate that an ABE is more likely to edit an A if a nearby A in the same DNA strand (the same sequencing read) is also edited. LD values are normalized from 0 to 1 in order to be independent of editing efficiency. Values and error bars reflect the mean and s.d. of normalized LD values from three independent biological replicates performed on different days.

FIGS. 14A and 14B show high-throughput DNA sequencing analysis of HEK293T cells treated with five late-stage ABE variants and an sgRNA targeting −198T in the promoter of HBG1 and HBG2. One representative replicate is shown of DNA sequences at the HBG1 (FIG. 14A) and HBG2 (FIG. 14B) promoter targets. ABE-mediated base editing installs a −198T→C mutation on the strand complementary to the one shown in the sequencing data tables. Data from untreated HEK293T cells are shown for comparison.

FIG. 15 shows base editing data using the base editor ABE 7.10 and 12 gRNAs that target the promotor region of HBG1/2. The identity of the gRNA target sequences are (1) SEQ ID NO: 259, (2) SEQ ID NO: 260 (3) SEQ ID NO: 266, (4) SEQ ID NO: 267, (5) SEQ ID NO: 268, (6) SEQ ID NO: 269, (7) SEQ ID NO: 270, (8) SEQ ID NO: 276, (9) SEQ ID NO: 277, (10) SEQ ID NO: 278, (11) SEQ ID NO: 279, and (12) SEQ ID NO: 280.

FIG. 16 shows HTS sequencing results and % indel of untreated HEK293T cells and HEK293T cells treated with ABE6.3, ABE7.8, ABE7.9, or ABE7.10 at 17 genomic sites with co-transfection of a corresponding sgRNA expression plasmid. One arbitrarily chosen replicate is shown; the data for all replicates is available from the NCBI sequencing read archive.

FIG. 17 shows activities of ABE7.8, ABE7.9, and ABE7.10 at the HEK2 on-target and off-target sites previously characterized for S. pyogenes Cas9 nuclease.

FIG. 18 shows activities of ABE7.8, ABE7.9, and ABE7.10 at the HEK3 site previously characterized for on-target and off-target modification by S. pyogenes Cas9 nuclease.

FIG. 19 shows activities of ABE7.8, ABE7.9, and ABE7.10 at the HEK4 site previously characterized for on-target and off-target modification by S. pyogenes Cas9 nuclease. Although HEK4 off-target site 3 showed appreciable indel formation upon ABE treatment, this locus also showed unusually high (89%) indel formation by Cas9 nuclease and was the only tested off-target site exhibiting indel formation upon treatment with Cas9 nickases. It is speculated that this locus is unusually fragile, and that indel formation here arises from simply nicking the site, rather than from ABE-mediated adenine deamination.

FIG. 20 shows primers used for generating sgRNA plasmids. The 20-nt target protospacer is shown in red. When a target DNA sequence did not start with a ‘G’, a ‘G’ was added to the 5′ end of the primer since the human U6 promoter prefers a ‘G’ at the transcription start site. The pFYF sgRNA plasmids described previously were used as a template for PCR amplification.

FIG. 21 shows primers used for generating bacterial TadA* libraries.

FIG. 22 shows starting constructs used for each round of TadA* mutagenesis and selection in E. coli. All plasmids contain an SC101 origin of replication, a β-lactamase gene for plasmid maintenance with carbenicillin, a P_(BAD) promoter driving TadA*-dCas9 expression, and a lac promoter driving sgRNA transcription. The architecture of the base editors used during bacterial selection is: TadA*-linker(16 aa)-dCas9.

FIG. 23 shows antibiotic selection plasmids and their corresponding E. coli antibiotic minimum inhibitory concentrations (MICs).

FIG. 24 shows primers used for mammalian cell genomic DNA amplification.

FIG. 25 shows 100-mer single-stranded oligonucleotide donor templates (ssODNs) used in HDR experiments.

DEFINITIONS

As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

The term “deaminase” or “deaminase domain” refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g. engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as, E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine

In some embodiments, the TadA deaminase is an N-terminal truncated TadA. In certain embodiments, the adenosine deaminase comprises the amino acid sequence:

(SEQ ID NO: 1) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD.

In some embodiments the TadA deaminase is a full-length E. coli TadA deaminase. For example, in certain embodiments, the adenosine deaminase comprises the amino acid sequence:

(SEQ ID NO: 84) MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNR VIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVM CAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILAD ECAALLSDFFRMRRQEIKAQKKAQSSTD

It should be appreciated, however, that additional adenosine deaminases useful in the present application would be apparent to the skilled artisan and are within the scope of this disclosure. For example, the adenosine deaminase may be a homolog of an ADAT. Exemplary ADAT homologs include, without limitation:

Staphylococcus aureus TadA: (SEQ ID NO: 8) MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRET LQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIP RVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN Bacillus subtilis TadA: (SEQ ID NO: 9) MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRS IAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVF GAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK KKKAARKNLSE Salmonella typhimurium (S. typhimurium) TadA: (SEQ ID NO: 371) MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHR VIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVM CAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRD ECATLLSDFFRMRRQEIKALKKADRAEGAGPAV Shewanella putrefaciens (S. putrefaciens) TadA: (SEQ ID NO: 372) MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTA HAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGA RDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK KALKLAQRAQQGIE Haemophilus influenzae F3031 (H. influenzae) TadA: (SEQ ID NO: 373) MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWN LSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK Caulobacter crescentus (C. crescentus) TadA: (SEQ ID NO: 374) MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGN GPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISH ARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLR GFFRARRKAKI Geobacter sulfurreducens (G. sulfurreducens) TadA: (SEQ ID NO: 375) MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHN LREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIIL ARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLS DFFRDLRRRKKAKATPALFIDERKVPPEP

The term “base editor (BE),” or “nucleobase editor (NBE)” refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenine (A) in DNA. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In some embodiments, the dCas9 domain of the fusion protein comprises a D10A and a H840A mutation of SEQ ID NO: 52, or a corresponding mutation in any of SEQ ID NOs: 108-357, which inactivates the nuclease activity of the Cas9 protein. In some embodiments, the fusion protein comprises a D10A mutation and comprises a histidine at residue 840 of SEQ ID NO: 52, or a corresponding mutation in any of SEQ ID NOs: 108-357, which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex. An example of a Cas9 nickase is shown in SEQ ID NO: 35.

The term “linker,” as used herein, refers to a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid-editing domain (e.g., an adenosine deaminase). In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein. In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 10, 37-40, 384-386, 685-688, or 800-801. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 10), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 37). In some embodiments, a linker comprises the amino acid sequence (SGGS)₂-SGSETPGTSESATPES-(SGGS)₂ (SEQ ID NO: 800), which may also be referred to as (SGGS)₂-XTEN-(SGGS)₂. In some embodiments, a linker comprises (SGGS)_(n)(SEQ ID NO: 37), (GGGS)_(n)(SEQ ID NO: 38), (GGGGS)_(n)(SEQ ID NO: 39), (G)_(n), (EAAAK)_(n)(SEQ ID NO: 40), (GGS)_(n), SGSETPGTSESATPES (SEQ ID NO: 10), (SGGS)_(n)-SGSETPGTSESATPES-(SGGS)_(n)(SEQ ID NO: 801) or (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^(th) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 4), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5), MKRTADGSEFEPKKKRKV (SEQ ID NO: 342), or KRTADGSEFEPKKKRKV (SEQ ID NO: 343).

The term “nucleic acid programmable DNA binding protein” or “napDNAbp” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, that guides the napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that has complementary to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNAbinding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically listed in this disclosure.

The term “Cas9” or “Cas9 domain” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (mc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO: 47 (nucleotide); SEQ ID NO: 48 (amino acid)).

(SEQ ID NO: 47) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG ATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGG TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT CTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGAT TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC TATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCT ATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA GAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCT AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT TTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCT ATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTC TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG AAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATT ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGG AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT AGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGG CCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTA AAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTA ATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCA GACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCG AAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAA TGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTG ATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCA ATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGA TAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGAC AACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAA ACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGA GAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAA AGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC ATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATAT CCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGT TCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAA AATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACA CTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCA AAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAG ACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAA GCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTG ATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAA GGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAAT TATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTA AAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATAT AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGG AGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATT TTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGAT AACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGA GATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATG CCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCA ATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCT TGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC GATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCC ATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGA CTGA (SEQ ID NO: 48) MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA LLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV MGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO:49 (nucleotide) and/or SEQ ID NO: 50 (amino acid):

(SEQ ID NO: 49) ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGG ATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGG TGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCC CTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAAC CGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAG AAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGT TTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCC CATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGAC CTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCA CTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAAC TGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCT ATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTC TAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGA AAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCA AATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAG ATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATC CTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTT ATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACAC TTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATA TTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGC GAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGG ATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGG CGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCA AAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTAC TATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAG AAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATA AAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAG AATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTA TTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGAT CTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGA CTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAG AAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATA ATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGA AGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGG AAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAG TTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAA AGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGAC TCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGG GGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCA AAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTC ATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAA TCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAA TAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACA AAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTAT CTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGAT TCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAG TGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGC GGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTA ACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTAT TAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATT CGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAG AAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATG CGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAA TACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGA CGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAG CCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATC ACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGA GAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTG CAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGA TAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCT TCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAG AAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAAC GATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGG CGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGC CGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGA ATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAA GATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGA CGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTG ATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAA CCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAA CCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAA TCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGG TGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACC ATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC AAGGCTGCAGGA (SEQ ID NO: 50) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, SEQ ID NO: 51 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 52 (amino acid).

(SEQ ID NO: 51) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG ATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGG TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT CTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC TATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCT ATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA AAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCT AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT TTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCT ATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTC TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG AAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATT ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGG AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT AGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGG CGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTA AAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTA ATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAA TCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAA TCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCA AAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAA GTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATC GGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGA GACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA ACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTAT CAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATT CGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCG AAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATG CCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAA TATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGA TGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG CAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGC GCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTA CAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGA CAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTT TTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCAC AATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAG CTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGC CGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGA ATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAA GATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGA TGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAA CCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAA TCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAA TCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGG TGACTGA (SEQ ID NO: 52) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1); Geobacillus stearothermophilus (NCBI Ref: NZ_CP008934.1); or Neisseria. meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism.

In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and an H840A mutation of SEQ ID NO: 52 or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the amino acid sequence of SEQ ID NO: 53 dCas9 (D10A and H840A):

(SEQ ID NO: 53) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLS ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain).

In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided in SEQ ID NO: 52, or at corresponding positions in any of the amino acid sequences provided in SEQ ID NOs: 108-357. Without wishing to be bound by any particular theory, the presence of the catalytic residue H840 maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A. Restoration of H840 (e.g., from A840 of a dCas9) does not result in the cleavage of the target strand containing the A. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand. A schematic representation of this process is shown in FIG. 1C. Briefly, and without wishing to be bound by any particular theory, the A of a A-T base pair can be deaminated to a inosine (I) by an adenosine deaminase, e.g., an engineered adenosine deaminase that deaminates an adenosine in DNA. Nicking the non-edited strand, having the T, facilitates removal of the T via mismatch repair mechanisms. A UGI domain or a catalytically inactive inosine-specific nuclease (dISN) may inhibit inosine-specific nucleases (e.g., sterically) thereby preventing removal of the inosine (I).

In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 (e.g., variants of SEQ ID NO: 53) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 10. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 53) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 53, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more.

In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all.

Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Geobacillus stearothermophilus (NCBI Ref: NZ_CP008934.1); or Neisseria. meningitidis (NCBI Ref: YP_002342100.1).

It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises the amino acid sequence (SEQ ID NO: 34). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid sequence (SEQ ID NO: 35). In some embodiments, the Cas9 protein is a nuclease active Cas9. In some embodiments, the nuclease active Cas9 comprises the amino acid sequence (SEQ ID NO: 36).

Exemplary Catalytically Inactive Cas9 (dCas9):

(SEQ ID NO: 34) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD Exemplary Cas9 Nickase (nCas9):

(SEQ ID NO: 35) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD Exemplary Catalytically Active Cas9:

(SEQ ID NO: 36) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD.

In some embodiments, Cas9 refers to a Cas9 from arehaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any one of SEQ ID NOs: 417-419. In some embodiments, the napDNAbp comprises an amino acid sequence of any one SEQ ID NOs: 417-419. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

CasX (uniprot.org/uniprot/FONN87; uniprot.org/uniprot/F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus  (strain HVE10/4) GN = SiH_0402 PE = 4 SV = 1 (SEQ ID NO: 417) MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG IVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGG FSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus  (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1 (SEQ ID NO: 418) MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLE VEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG IVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGG FSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG CasY (ncbi.nlm.nih.gov/protein/APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium] (SEQ ID NO: 419) MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPRE IVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFS YTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRA NGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQK KLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKL KEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELK KAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDIN GKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQE ALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNF YGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKD FFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQS RSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEE YIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLE GRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHE FQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVL YVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTV ALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEIT GDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESL VHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE IDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQ ELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKM RGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nucleobase editor may refer to the amount of the nucleobase editor that is sufficient to induce mutation of a target site specifically bound mutated by the nucleobase editor. In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a fusion protein comprising a nucleic acid programmable DNA binding protein and a deaminase domain (e.g., an adenosine deaminase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nucleobase editor, a deaminase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.

The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term “promoter” as used herein refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors.

The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^(th) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target), referred to as a guide sequence; and (2) a domain that binds a Cas9 protein. In some embodiments, domain (1) shares homology with a sequence in the promoter of the HBG1 and/or the HBG2 gene. In some embodiments, domain (1) shares homology with the sequence 5′-GTGGGGAAGGGGCCCCCAAGAGG-3′ (SEQ ID NO: 2). In some embodiments, domain (1) shares homology with a sequence in the HFE gene. In some embodiments, domain (1) shares homology with the sequence 5′-TATACGTACCAGGTGGAGCACCCAGG-3′ (SEQ ID NO: 3). In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and U.S. Provisional Patent Application, U.S. Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference.

Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

The term “target site” refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase, (e.g., a dCas9-adenosine deaminase fusion protein provided herein).

The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

DETAILED DESCRIPTION OF THE INVENTION

Some aspects of this disclosure relate to proteins that deaminate the nucleobase adenine. This disclosure provides adenosine deaminase proteins that are capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA). For example, the adenosine deaminases provided herein are capable of deaminating adenine of a deoxyadenosine residue of DNA. Other aspects of the disclosure provide fusion proteins that comprise an adenosine deaminase (e.g., an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein) and a domain (e.g., a Cas9 or a Cpf1 protein) capable of binding to a specific nucleotide sequence. The deamination of an adenosine by an adenosine deaminase can lead to a point mutation, this process is referred to herein as nucleic acid editing. For example, the adenosine may be converted to an inosine residue, which typically base pairs with a cytosine residue. Such fusion proteins are useful inter alia for targeted editing of nucleic acid sequences. Such fusion proteins may be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject. As an example, diseases that can be treated by making an A to G, or a T to C mutation, may be treated using the nucleobase editors provided herein. Without wishing to be bound by any particular theory certain anemias, such as sickle cell anemia, may be treated by inducing expression of hemoglobin, such as fetal hemoglobin, which is typically silenced in adults. As one example, mutating −198T to C in the promoter driving HBG1 and HB G2 gene expression results in increased expression of HBG1 and HBG2.

Another example, a class of disorders that results from a G to A mutation in a gene is iron storage disorders, where the HFE gene comprises a G to A mutation that results in expression of a C282Y mutant HFE protein. Thus, the adenosine base editors described herein may be utilized for the targeted editing of such G to A mutations (e.g., targeted genome editing). The invention provides deaminases, fusion proteins, nucleic acids, vectors, cells, compositions, methods, kits, systems, etc. that utilize the deaminases and nucleobase editors.

In some embodiments, the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein. In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A. Mutation of the catalytic residue (e.g., D10 to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand.

Adenosine Deaminases

Some aspects of the disclosure provide adenosine deaminases. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1, 64-84, 420-437, 672-684, 802-805, or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identiy plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 1, 64-84, 420-437, 672-684, 802-805, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 1, 64-84, 420-437, 672-684, 802-805, or any of the adenosine deaminases provided herein.

In some embodiments, the adenosine deaminase comprises a D108X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. It should be appreciated, however, that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein.

In some embodiments, the adenosine deaminse comprises an A106X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises a E155X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155D, E155G, or E155V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises a D147X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

It should be appreciated that any of the mutations provided herein (e.g., based on the ecTadA amino acid sequence of SEQ ID NO: 1) may be introduced into other adenosine deaminases, such as S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan how to identify amino acid residues from other adenosine deaminases that are homologous to the mutated residues in ecTadA. Thus, any of the mutations identified in ecTadA may be made in other adenosine deaminases that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein may be made individually or in any combination in ecTadA or another adenosine deaminase. For example, an adenosine deaminase may contain a D108N, a A106V, a E155V, and/or a D147Y mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a “;”) in ecTadA SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V, E55V, and D147Y; and D108N, A106V, E55V, and D147Y. It should be appreciated, however, that any combination of corresponding mutations provided herein may be made in an adenosine deaminase (e.g., ecTadA). In some embodiments, an adenosine deaminase comprises one or more of the mutations shown in FIG. 7 , which identifies individual mutations and combinations of mutations made in ecTadA. In some embodiments, an adenosine deaminase comprises any mutation or combination of mutations provided herein.

In some embodiments, the adenosine deaminse comprises an L84X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an H123X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an I156X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I156F mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K1605 in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an A142X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G, mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an H36X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an N37X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T, or N37S mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a N37S mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an P48X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an P48T, P48S, P48A, or P48L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48T mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48A mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an R51X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H, or R51L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R51L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an S146X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R, or S146C mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a S146C mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an K157X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an W23X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R, or W23L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an R152X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P, or R52H mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152H mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an R26X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R26G mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an I49X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a I49V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an N72X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a N72D mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an S97X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a S97C mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an G125X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a G125A mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminse comprises an K161X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K161T mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one or more of a W23X, H36X, N37X, P48X, I49X, R51X, N72X, L84X, S97X, A106X, D108X, H123X, G125X, A142X, S146X, D147X, R152X, E155X, I156X, K157X, and/or K161X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of W23L, W23R, H36L, P48S, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and/or K157N mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations provided in FIG. 7 corresponding to SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one or two mutations selected from A106X and D108X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one or two mutations selected from A106V and D108N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, or four mutations selected from A106X, D108X, D147X, and E155X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, or four mutations selected from A106V, D108N, D147Y, and E155V in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a A106V, D108N, D147Y, and E155V mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, or seven mutations selected from L84X, A106X, D108X, H123X, D147X, E155X, and I156X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, or seven mutations selected from L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, or eleven mutations selected from H36X, R51X, L84X, A106X, D108X, H123X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, or eleven mutations selected from H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminse comprises or consists of a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, S146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminse comprises or consists of a W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and K157N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one or more of the mutations provided in FIG. 7 corresponding to SEQ ID NO: 1, or one or more of the corresponding mutations in another deaminase. In some embodiments, the adenosine deaminase comprises or consists of a variant of SEQ ID NO: 1 provided in FIG. 7 , or the corresponding variant in another adenosine deaminse.

It should be appreciated that the adenosine deaminase (e.g., a first or second adenosine deaminase) may comprise one or more of the mutations provided in any of the adenosine deaminases (e.g., ecTadA adenosine deaminases) shown in FIG. 7 . In some embodiments, the adenosine deaminase comprises the combination of mutations of any of the adenosine deaminases (e.g., ecTadA adenosine deaminases) shown in FIG. 7 . For example, the adenosine deaminase may comprise the mutations W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N (relative to SEQ ID NO: 1), which is shown as ABE7.10 in FIG. 7 . In some embodiments, the adenosine deaminase may comprise the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N (relative to SEQ ID NO: 1). In some embodiments, the adenosine deaminase comprises any of the following combination of mutations relative to SEQ ID NO:1, where each mutation of a combination is separated by a “_” and each combination of mutations is between parentheses: (A106V_D108N), (R107C_D108N), (H8Y_D108N_S127S_D147Y_Q154H), (H8Y_R24W_D108N_N127S_D147Y_E155V), (D108N_D147Y_E155V), (H8Y_D108N_S127S), (H8Y_D108N_N127S_D147Y_Q154H), (A106V_D108N_D147Y_E155V), (D108Q_D147Y_E155V), (D108M_D147Y_E155V), (D108L_D147Y_E155V), (D108K_D147Y_E155V), (D108I_D147Y_E155V), (D108F_D147Y_E155V), (A106V_D108N_D147Y), (A106V_D108M_D147Y_E155V), (E59A_A106V_D108N_D147Y_E155V), (E59A cat dead_A106V_D108N_D147Y_E155V), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D103A_D014N), (G22P_D103A_D104N), (G22P_D103A_D104N_S138A), (D103A_D104N_S138A), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F), (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F), (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156F), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (A106V_D108N_A142N_D147Y_E155V), (R26G_A106V_D108N_A142N_D147Y_E155V), (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V), (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V), (E25D_R26G_A106V_D108N_A142N_D147Y_E155V), (A106V_R107K_D108N_A142N_D147Y_E155V), (A106V_D108N_A142N_A143G_D147Y_E155V), (A106V_D108N_A142N_A143L_D147Y_E155V), (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N), (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F), (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T), (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F), (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F), (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F), (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E), (H36L_G67V_L84F_A106V_D108N_H123Y_S146T D147Y_E155V_I156F), (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F), (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F), (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F), (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F), (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T), (L84F_A106V_D108N_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E), (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F), (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (P48S_A142N), (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N), (P48T_I49V_A142N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N), (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N).

-   In some embodiments, the adenosine deaminase comprises an amino acid     sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95,     98%, 99%, or 99.5% identical to any one of SEQ ID NOs: 1, 64-84,     420-437, 672-684, 802-805, or any of the adenosine deaminases     provided herein. In some embodiments, the adenosine deaminase     comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9,     10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26,     27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,     44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of     the amino acid sequences set forth in SEQ ID NOs: 1, 64-84, 420-437,     672-684, 802-805, or any of the adenosine deaminases provided     herein. In some embodiments, the adenosine deaminase comprises an     amino acid sequence that has at least 5, at least 10, at least 15,     at least 20, at least 25, at least 30, at least 35, at least 40, at     least 45, at least 50, at least 60, at least 70, at least 80, at     least 90, at least 100, at least 110, at least 120, at least 130, at     least 140, at least 150, at least 160, or at least 166, identical     contiguous amino acid residues as compared to any one of the amino     acid sequences set forth in SEQ ID NOs: 1, 64-84, 420-437, 672-684,     802-805, or any of the adenosine deaminases provided herein. In some     embodiments, the adenosine deaminase comprises the amino acid     sequence of any one of SEQ ID NOs: 1, 64-84, 420-437, 672-684,     802-805, or any of the adenosine deaminases provided herein. In some     embodiments, the adenosine deaminase consists of the amino acid     sequence of any one of SEQ ID NOs: 1, 64-84, 420-437, 672-684,     802-805, or any of the adenosine deaminases provided herein. The     ecTadA sequences provided below are from ecTadA (SEQ ID NO: 1),     absent the N-terminal methionine (M). The saTadA sequences provided     below are from saTadA (SEQ ID NO: 8), absent the N-terminal     methionine (M). For clarity, the amino acid numbering scheme used to     identify the various amino acid mutations is derived from ecTadA     (SEQ ID NO: 1) for E. coli TadA and saTadA (SEQ ID NO: 8) for S.     aureus TadA. Amino acid mutations, relative to SEQ ID NO: 1 (ecTadA)     or SEQ ID NO: 8 (saTadA), are indicated by underlining.

ecTadA  (SEQ ID NO: 64) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (D108N)  (SEQ ID NO: 65) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (D108G)  (SEQ ID NO: 66) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARGAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (D108V)  (SEQ ID NO: 67) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARVAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (H8Y, D108N, and N127S)  (SEQ ID NO: 68) SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (H8Y, D108N, N1275, and E155D)  (SEQ ID NO: 69) SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRM RRQDIKAQKKAQSSTD ecTadA (H8Y, D108N, N1275, and E155G)  (SEQ ID NO: 70) SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRM RRQGIKAQKKAQSSTD ecTadA (H8Y, D108N, N1275, and E155V)  (SEQ ID NO: 71) SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRM RRQVIKAQKKAQSSTD ecTadA (A106V, D108N, D147Y, and E155V)  (SEQ ID NO: 72) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSYFFRM RRQVIKAQKKAQSSTD ecTadA (L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 73) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (S2A, I49F, A106V, D108N, D147Y, E155V)  (SEQ ID NO: 74) AEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPFGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSYFFRM RRQVIKAQKKAQSSTD ecTadA (H8Y, A106T, D108N, N1275, K1605)  (SEQ ID NO: 75) SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGTRNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRM RRQEIKAQSKAQSSTD ecTadA (R26G, L84F, A106V, R107H, D108N, H123Y, A142N, A143D, D147Y, E155V, I156F)  (SEQ ID NO: 76) SEVEFSHEYWMRHALTLAKRAWDEGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRM RRQVFKAQKKAQSSTD ecTadA (E25G, R26G, L84F, A106V, R107H, D108N, H123Y, A142N, A143D, D147Y, E155V, I156F)  (SEQ ID NO: 77) SEVEFSHEYWMRHALTLAKRAWDGGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRM RRQVFKAQKKAQSSTD ecTadA (E25D, R26G, L84F, A106V, R107K, D108N, H123Y, A142N, A143G, D147Y, E155V, I156F)  (SEQ ID NO: 78) SEVEFSHEYWMRHALTLAKRAWDDGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVKNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNGLLSYFFRM RRQVFKAQKKAQSSTD ecTadA (R26Q, L84F, A106V, D108N, H123Y, A142N, D147Y, E155V, I156F)  (SEQ ID NO: 79) SEVEFSHEYWMRHALTLAKRAWDEQEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (E25M, R26G, L84F, A106V, R107P, D108N, H123Y, A142N, A143D, D147Y, E155V, I156F)  (SEQ ID NO: 80) SEVEFSHEYWMRHALTLAKRAWDMGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVPNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRM RRQVFKAQKKAQSSTD ecTadA (R26C, L84F, A106V, R107H, D108N, H123Y, A142N , D147Y, E155V, I156F)  (SEQ ID NO: 81) SEVEFSHEYWMRHALTLAKRAWDECEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (L84F, A106V , D108N, H123Y, A142N, A143L, D147Y, E155V, I156F)  (SEQ ID NO: 82) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNLLLSYFFRM RRQVFKAQKKAQSSTD ecTadA (R26G, L84F, A106V, D108N, H123Y, A142N , D147Y, E155V, I156F)  (SEQ ID NO: 83) SEVEFSHEYWMRHALTLAKRAWDEGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (E25A, R26G, L84F, A106V, R107N, D108N, H123Y, A142N, A143E, D147Y, E155V, I156F)  (SEQ ID NO: 420) SEVEFSHEYWMRHALTLAKRAWDAGEVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVNNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNELLSYFFRM RRQVFKAQKKAQSSTD ecTadA (L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 421) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (N37T, P48T, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 422) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHTNRVIGEGWNRTIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (N375, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 423) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHSNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (H36L, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 424) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (L84F, A106V, D108N, H123Y, 5146R, D147Y, E155V, I156F)  (SEQ ID NO: 425) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLRYFFRM RRQVFKAQKKAQSSTD ecTadA (H36L, P48L, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 426) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRLIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (H36L, L84F, A106V, D108N, H123Y, D147Y, E155V, K57N, I156F)  (SEQ ID NO: 427) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFNAQKKAQSSTD ecTadA (H36L, L84F, A106V, D108N, H123Y, 5146C, D147Y, E155V, I156F)  (SEQ ID NO: 428) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM RRQVFKAQKKAQSSTD ecTadA (L84F, A106V, D108N, H123Y, 5146R, D147Y, E155V, I156F)  (SEQ ID NO: 429) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLRYFFRM RRQVFKAQKKAQSSTD ecTadA (N375, R51H, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 430) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHSNRVIGEGWNRPIGH HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (R51L, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 431) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFNAQKKAQSSTD ecTadA (R51H, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 432) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGH HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFNAQKKAQSSTD ecTadA (P48S)  (SEQ ID NO: 672) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRSIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (P48T)  (SEQ ID NO: 673) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRTIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (P48A)  (SEQ ID NO: 674) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (A142N)  (SEQ ID NO: 675) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECNALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (W23R)  (SEQ ID NO: 676) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (W23L)  (SEQ ID NO: 677) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM RRQEIKAQKKAQSSTD ecTadA (R152P)  (SEQ ID NO: 678) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM PRQEIKAQKKAQSSTD ecTadA (R152H)  (SEQ ID NO: 679) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM HRQEIKAQKKAQSSTD ecTadA (L84F, A106V, D108N, H123Y, D147Y, E155V, I156F)  (SEQ ID NO: 680) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRM RRQVFKAQKKAQSSTD ecTadA (H36L, R51L, L84F, A106V, D108N, H123Y, 5146C, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 681) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM RRQVFNAQKKAQSSTD ecTadA (H36L, P48S, R51L, L84F, A106V, D108N, H123Y, 5146C, D147Y, E155V, I156F,  K157N)  (SEQ ID NO: 682) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM RRQVFNAQKKAQSSTD ecTadA (H36L, P48A, R51L, L84F, A106V, D108N, H123Y, 5146C, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 683) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM RRQVFNAQKKAQSSTD ecTadA (W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, 5146C, D147Y, R152P, E155V, I156F, K157N)  (SEQ ID NO: 684) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM PRQVFNAQKKAQSSTD ecTadA (H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, 5146C, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 802) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRM RRQVFNAQKKAQSSTD ecTadA (W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, K157N)  (SEQ ID NO: 803) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRM RRQVFNAQKKAQSSTD ecTadA (W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, 5146C, D147Y, R152P, E155V, I156F, K157N)  (SEQ ID NO: 804) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRM PRQVFNAQKKAQSSTD ecTadA (W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, 5146C, D147Y, R152P, E155V, I156F, K157N)  (SEQ ID NO: 805) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM PRQVFNAQKKAQSSTD Cas9 Domains of Nucleobase Editors

In some aspects, a nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences as set forth in SEQ ID NOs: 108-357. In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 108-357. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 108-357. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 108-357.

In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth in SEQ ID NO: 52, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth in SEQ ID NO: 52, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 54 (Cloning vector pPlatTET-gRNA2, Accession No. BAV54124).

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD(SEQ ID NO: 54; see, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference). 

Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). In some embodiments the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequences that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 108-357. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 108-357.

In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840 of SEQ ID NO: 52, or a mutation in any of SEQ ID NOs: 108-357. As one example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 35. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired to a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10 of SEQ ID NO: 52, or a corresponding mutation in any of SEQ ID NOs: 108-357. In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.

Cas9 Domains with Reduced PAM Exclusivity

Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises the amino acid sequence SEQ ID NO: 55. In some embodiments, the SaCas9 comprises a N579X mutation of SEQ ID NO: 55, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation of SEQ ID NO: 55, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357.

In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT PAM sequence, where N=A, T, C, or G, and R=A or G. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutation of SEQ ID NO: 55, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation of SEQ ID NO: 55, or one or more corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation of SEQ ID NO: 55, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 108-357.

In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 55-57. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 55-57. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 55-57.

Exemplary SaCas9 Sequence

(SEQ ID NO: 55) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF NYEVDHIIPRSVSFDNSFNNKVLVKQEE N SKKGNRTPFQYLSSSDSKISY ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG

Residue N579 of SEQ ID NO: 55, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase.

Exemplary SaCas9n Sequence

(SEQ ID NO: 56) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF NYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISY ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG.

Residue A579 of SEQ ID NO: 56, which can be mutated from N579 of SEQ ID NO: 55 to yield a SaCas9 nickase, is underlined and in bold.

Exemplary SaKKH Cas9

(SEQ ID NO: 57) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF NYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISY ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK KLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY REYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG.

Residue A579 of SEQ ID NO: 57, which can be mutated from N579 of SEQ ID NO: 55 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 of SEQ ID NO: 57, which can be mutated from E781, N967, and R1014 of SEQ ID NO: 55 to yield a SaKKH Cas9 are underlined and in italics.

In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises the amino acid sequence SEQ ID NO: 58. In some embodiments, the SpCas9 comprises a D9X mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134E, R1334Q, and T1336R mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35. In some embodiments, the SpCas9 domain comprises a D1134E, a R1334Q, and a T1336R mutation of SEQ ID NO: 58, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 108-35. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35. In some embodiments, the SpCas9 domain comprises a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 58, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 108-35. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a G1217X, a R1334X, and a T1336X mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 58, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-35. In some embodiments, the SpCas9 domain comprises a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 58, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 108-35.

In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 58-62. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 58-62. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 58-62.

Exemplary SpCas9

(SEQ ID NO: 58) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD Exemplary SpCas9n

(SEQ ID NO: 59) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD Exemplary SpEQR Cas9

(SEQ ID NO: 60) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF E SPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD

Residues E1134, Q1334, and R1336 of SEQ ID NO: 60, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 58 to yield a SpEQR Cas9, are underlined and in bold.

Exemplary SpVQR Cas9

(SEQ ID NO: 61) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD

Residues V1134, Q1334, and R1336 of SEQ ID NO: 61, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 58 to yield a SpVQR Cas9, are underlined and in bold.

Exemplary SpVRER Cas9

(SEQ ID NO: 62) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD

Residues V1134, R1217, Q1334, and R1336 of SEQ ID NO: 62, which can be mutated from D1134, G1217, R1334, and T1336 of SEQ ID NO: 58 to yield a SpVRER Cas9, are underlined and in bold.

High Fidelity Cas9 Domains

Some aspects of the disclosure provide high fidelity Cas9 domains of the nucleobase editors provided herein. In some embodiments, high fidelity Cas9 domains are engineered Cas9 domains comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA, as compared to a corresponding wild-type Cas9 domain. Without wishing to be bound by any particular theory, high fidelity Cas9 domains that have decreased electrostatic interactions with the sugar-phosphate backbone of DNA may have less off-target effects. In some embodiments, the Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more.

In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of N497X, R661X, Q695X, and/or Q926X mutation of the amino acid sequence provided in SEQ ID NO: 52, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of N497A, R661A, Q695A, and/or Q926A mutation of the amino acid sequence provided in SEQ ID NO: 52, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357. In some embodiments, the Cas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 52, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 108-357. In some embodiments, the Cas9 domain (e.g., of any of the fusion proteins provided herein) comprises the amino acid sequence as set forth in SEQ ID NO: 62. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each are incorporated herein by reference.

It should be appreciated that any of the base editors provided herein, for example, any of the adenosine deaminase base editors provided herein, may be converted into high fidelity base editors by modifying the Cas9 domain as described herein to generate high fidelity base editors, for example, a high fidelity adenosine base editor. In some embodiments, the high fidelity Cas9 domain is a dCas9 domain. In some embodiments, the high fidelity Cas9 domain is a nCas9 domain.

High Fidelity Cas9 Domain where Mutations Relative to Cas9 of SEQ ID NO: 10 are Shown in Bold and Underlines

(SEQ ID NO: 63) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT A FDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD Nucleic Acid Programmable DNA Binding Proteins

Some aspects of the disclosure provide nucleic acid programmable DNA binding proteins, which may be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. Nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. One example of an nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, C2c1, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (C2c1, C2c2, and C2c3) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference.

Fusion Proteins Comprising a Nuclease Programmable DNA Binding Protein and an Adenosine Deaminase

Some aspects of the disclosure provide fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase. In some embodiments, any of the fusion proteins provided herein are base editors. In some embodiments, the napDNAbp is a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a C2c1 domain, a C2c2 domain, aC2c3 domain, or an Argonaute domain. In some embodiments, the napDNAbp is any napDNAbp provided herein. Some aspects of the disclosure provide fusion proteins comprising a Cas9 domain and an adenosine deaminase. The Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the adenosine deaminases provided herein. In some embodiments, the fusion protein comprises the structure:

NH₂-[adenosine deaminase]-[napDNAbp]-COOH; or

NH₂-[napDNAbp]-[adenosine deaminase]-COOH

In some embodiments, the fusion proteins comprising an adenosine deaminase and a napDNAbp (e.g., Cas9 domain) do not include a linker sequence. In some embodiments, a linker is present between the adenosine deaminase domain and the napDNAbp. In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker. In some embodiments, the adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the adenosine deaminase and the napDNAbp are fused via any of the linkers provided below in the section entitled “Linkers”. In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that comprises between 1 and and 200 amino acids. In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that comprises 3, 4, 16, 24, 32, 64, 100, or 104 amino acids in length. In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that comprises the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 10), SGGS (SEQ ID NO: 37), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 384), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 385), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 386). In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 10), which may also be referred to as the XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 685). In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS)₂-SGSETPGTSESATPES-(SGGS)₂ (SEQ ID NO: 800), which may also be referred to as (SGGS)₂-XTEN-(SGGS)₂. In some embodiments, the linker comprises the amino acid sequence (SGGS)_(n)-SGSETPGTSESATPES-(SGGS)_(n) (SEQ ID NO: 801), wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 686). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 687). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 688).

Fusion Proteins Comprising a Nuclear Localization Sequence (NLS)

In some embodiments, the fusion proteins provided herein further comprise one or more nuclear targeting sequences, for example, a nuclear localization sequence (NLS). In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, any of the fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the IBR (e.g., dISN). In some embodiments, the NLS is fused to the C-terminus of the IBR (e.g., dISN). In some embodiments, the NLS is fused to the N-terminus of the napDNAbp. In some embodiments, the NLS is fused to the C-terminus of the napDNAbp. In some embodiments, the NLS is fused to the N-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 4), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5), MKRTADGSEFEPKKKRKV (SEQ ID NO: 342), or KRTADGSEFEPKKKRKV (SEQ ID NO: 343).

In some embodiments, the general architecture of exemplary fusion proteins with an adenosine deaminase and a napDNAbp comprises any one of the following structures, where NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH₂ is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. Fusion proteins comprising an adenosine deaminase, a napDNAbp, and a NLS:

NH₂-[NLS]-[adenosine deaminase]-[napDNAbp]-COOH;

NH₂-[adenosine deaminase]-[NLS]-[napDNAbp]-COOH;

NH₂-[adenosine deaminase]-[napDNAbp]-[NLS]-COOH;

NH₂-[NLS]-[napDNAbp]-[adenosine deaminase]-COOH;

NH₂-[napDNAbp]-[NLS]-[adenosine deaminase]-COOH; and

NH₂-[napDNAbp]-[adenosine deaminase]-[NLS]-COOH.

In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., adenosine deaminase, napDNAbp, and/or NLS). In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.

Some aspects of the disclosure provide fusion proteins that comprise a nucleic acid programmable DNA binding protein (napDNAbp) and at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example to deaminate adenine. In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminase domains. In some embodiments, any of the fusion proteins provided herein comprise two adenosine deaminases. In some embodiments, any of the fusion proteins provided herein contain only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein, and the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase. As one example, the fusion protein may comprise a first adenosine deaminase and a second adenosine deaminase that both comprise the amino acid sequence of SEQ ID NO: 72, which contains a A106V, D108N, D147Y, and E155V mutation from ecTadA (SEQ ID NO: 1). In some embodiments, the fusion protein may comprise a first adenosine deaminase that comprises the amino acid sequence of SEQ ID NO: 682, which contains a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N mutation from SEQ ID NO: 1, and a second adenosine deaminase domain that comprises the amino amino acid sequence of wild-type ecTadA (SEQ ID NO: 1). In some embodiments, the fusion protein may comprise a first adenosine deaminase that comprises the amino acid sequence of SEQ ID NO: 802, which contains a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N mutation from SEQ ID NO: 1, and a second adenosine deaminase domain that comprises the amino amino acid sequence of wild-type ecTadA (SEQ ID NO: 1). In some embodiments, the fusion protein may comprise a first adenosine deaminase that comprises the amino acid sequence of SEQ ID NO: 803, which contains a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N mutation from SEQ ID NO: 1, and a second adenosine deaminase domain that comprises the amino amino acid sequence of wild-type ecTadA (SEQ ID NO: 1). In some embodiments, the fusion protein may comprise a first adenosine deaminase that comprises the amino acid sequence of SEQ ID NO: 804, which contains a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and K157N mutation from SEQ ID NO: 1, and a second adenosine deaminase domain that comprises the amino amino acid sequence of wild-type ecTadA (SEQ ID NO: 1). In some embodiments, the fusion protein may comprise a first adenosine deaminase that comprises the amino acid sequence of SEQ ID NO: 805, which contains a W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N mutation from SEQ ID NO: 1, and a second adenosine deaminase domain that comprises the amino amino acid sequence of wild-type ecTadA (SEQ ID NO: 1). Additional fusion protein constructs comprising two adenosine deaminase domains are illustrated in FIG. 7 .

In some embodiments, the fusion protein comprises two adenosine deaminases (e.g., a first adenosine deaminase and a second adenosine deaminase). In some embodiments, the fusion protein comprises a first adenosine deaminase and a second adenosine deaminase. In some embodiments, the first adenosine deaminase is N-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase is C-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase and the second deaminase are fused directly or via a linker. In some embodiments, the linker is any of the linkers provided herein, for example, any of the linkers described in the “Linkers” section. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 10, 37-40, 384-386, 685-688, or 800-801. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS)₂-SGSETPGTSESATPES-(SGGS)₂ (SEQ ID NO: 800), which may also be referred to as (SGGS)₂-XTEN-(SGGS)₂. In some embodiments, the linker comprises the amino acid sequence (SGGS)_(n)-SGSETPGTSESATPES-(SGGS)_(n) (SEQ ID NO: 801), wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the first adenosine deaminase is the same as the second adenosine deaminase. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are any of the adenosine deaminases described herein. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase is any of the adenosine deaminases provided herein but is not identical to the first adenosine deaminase. In some embodiments, the first adenosine deaminase is an ecTadA adenosine deaminase. In some embodiments, the first adenosine deaminase comprises an amino acid sequence that is at least least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1, 64-84, 420-437, 672-684, or to any of the adenosine deaminases provided herein. In some embodiments, the first adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second adenosine deaminase comprises an amino acid sequence that is at least least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1, 64-84, 420-437, 672-684, or to any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first adenosine deaminase and the second adenosine deaminase of the fusion protein comprise the mutations in ecTadA (SEQ ID NO: 1), or corresponding mutations in another adenosine deaminase, as shown in any one of the constructs provided in FIG. 19 (e.g., pNMG-371, pNMG-477, pNMG-576, pNMG-586, and pNMG-616). In some embodiments, the fusion protein comprises the two adenosine deaminases (e.g., a first adenosine deaminase and a second adenosine deaminase) of any one of the constructs (e.g., pNMG-371, pNMG-477, pNMG-576, pNMG-586, and pNMG-616) in FIG. 19 .

In some embodiments, the general architecture of exemplary fusion proteins with a first adenosine deaminase, a second adenosine deaminase, and a napDNAbp comprises any one of the following structures, where NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH₂ is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

Fusion proteins comprising a first adenosine deaminase, a second adenosine deaminase, and a napDNAbp. NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH₂-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH₂-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-COOH; NH₂-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-COOH; NH₂-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-COOH;

In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., first adenosine deaminase, second adenosine deaminase, and/or napDNAbp). In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.

Fusion proteins comprising a first adenosine deaminase, a second adenosine deaminase, a napDNAbp, and an NLS. NH₂-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-[NLS]-COOH; NH₂-[NLS]-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH₂-[first adenosine deaminase]-[NLS]-[napDNAbp]-[second adenosine deaminase]-COOH; NH₂-[first adenosine deaminase]-[napDNAbp]-[NLS]-[second adenosine deaminase]-COOH; NH₂-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-[NLS]-COOH; NH₂-[NLS]-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH₂-[napDNAbp]-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH₂-[napDNAbp]-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-COOH; NH₂-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-[NLS]-COOH; NH₂-[NLS]-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-COOH; NH₂-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-[napDNAbp]-COOH; NH₂-[second adenosine deaminase]-[first adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH₂-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-[NLS]-COOH; NH₂-[NLS]-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-COOH; NH₂-[second adenosine deaminase]-[NLS]-[napDNAbp]-[first adenosine deaminase]-COOH; NH₂-[second adenosine deaminase]-[napDNAbp]-[NLS]-[first adenosine deaminase]-COOH; NH₂-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-[NLS]-COOH; NH₂-[NLS]-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-COOH; NH₂-[napDNAbp]-[NLS]-[second adenosine deaminase]-[first adenosine deaminase]-COOH; NH₂-[napDNAbp]-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-COOH; NH₂-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-[NLS]-COOH;

In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., first adenosine deaminase, second adenosine deaminase, napDNAbp, and/or NLS). In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.

It should be appreciated that the fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein may comprise cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

Linkers

In certain embodiments, linkers may be used to link any of the protein or protein domains described herein. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 10), which may also be referred to as the XTEN linker. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS)₂-SGSETPGTSESATPES-(SGGS)₂ (SEQ ID NO: 800), which may also be referred to as (SGGS)₂-XTEN-(SGGS)₂. In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 37). In some embodiments, a linker comprises (SGGS)_(n) (SEQ ID NO: 37), (GGGS)_(n) (SEQ ID NO: 38), (GGGGS)_(n) (SEQ ID NO: 39), (G)_(n), (EAAAK)_(n) (SEQ ID NO: 40), (SGGS)_(n)-SGSETPGTSESATPES-(SGGS)_(n) (SEQ ID NO: 801), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 10), or (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, a linker comprises SGSETPGTSESATPES (SEQ ID NO: 10), and SGGS (SEQ ID NO: 37). In some embodiments, a linker comprises SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 384). In some embodiments, a linker comprises SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 385). In some embodiments, a linker comprises GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 386). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 685). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 686). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 687). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 688). It should be appreciated that any of the linkers provided herein may be used to link a first adenosine deaminase and a second adenosine deaminase; an adenosine deaminase (e.g., a first or a second adenosine deaminase) and a napDNAbp; a napDNAbp and an NLS; or an adenosine deaminase (e.g., a first or a second adenosine deaminase) and an NLS.

In some embodiments, any of the fusion proteins provided herein, comprise an adenosine deaminase and a napDNAbp that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise a first adenosine deaminase and a second adenosine deaminase that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise an NLS, which may be fused to an adenosine deaminase (e.g., a first and/or a second adenosine deaminase), a nucleic acid programmable DNA binding protein (napDNAbp), and or an inhibitor of base repair (IBR). Various linker lengths and flexibilities between an adenosine deaminase (e.g., an engineered ecTadA) and a napDNAbp (e.g., a Cas9 domain), and/or between a first adenosine deaminase and a second adenosine deaminase can be employed (e.g., ranging from very flexible linkers of the form (GGGGS)_(n) (SEQ ID NO: 38), (GGGGS)_(n) (SEQ ID NO: 39), and (G)_(n) to more rigid linkers of the form (EAAAK)_(n) (SEQ ID NO: 40), (SGGS)_(n) (SEQ ID NO: 37), SGSETPGTSESATPES (SEQ ID NO: 10) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)_(n)) in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1, 3, or 7. In some embodiments, the adenosine deaminase and the napDNAbp, and/or the first adenosine deaminase and the second adenosine deaminase of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 10), SGGS (SEQ ID NO: 37), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 384), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 385), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 386). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 685). In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS)₂-SGSETPGTSESATPES-(SGGS)₂ (SEQ ID NO: 800), which may also be referred to as (SGGS)₂-XTEN-(SGGS)₂. In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 686). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 687). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 688).

Some aspects of the disclosure provide fusion proteins comprising a Cas9 domain and an adenosine deaminase. Exemplary fusion proteins include, without limitation, the following fusion proteins (for the purposes of clarity, the adenosine deaminase domain is shown in Bold; mutations of the ecTadA deaminase domain are shown in Bold underlining; the XTEN linker is shown in italics; the UGI/AAG/EndoV domains are shown in Bold italics; and NLS is shown in underlined italics):

ecTadA(wt)-XTEN-nCas9-NLS: (SEQ ID NO: 11) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGSETPGTSESATPES DKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGS PKKKRKV ecTadA(D108N)-XTEN-nCas9-NLS: (mammalian construct, active on DNA, A to G editing):

(SEQ ID NO: 12) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGAR N AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGSETPGTSESATPESDKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGS PKKKRKV ecTadA(D108G)-XTEN-nCas9-NLS: (mammalian construct, active on DNA, A to G editing):

(SEQ ID NO: 13) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGAR G AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGSETPGTSESATPES DKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGS PKKKRKV ecTadA(D108V)-XTEN-nCas9-NLS: (mammalian construct, active on DNA, A to G editing):

(SEQ ID NO: 14) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGAR V AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGSETPGTSESATPES DKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGS PKKKRKV Variant resulting from first round of evolution (in bacteria) ecTadA(H8Y_D108N_N127S)-XTEN-dCas9:

(SEQ ID NO: 27) MSEVEFS Y EYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGSETPGTSESATPES DKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Enriched variants from second round of evolution (in bacteria) ecTadA (H8Y_D108N_N127S_E155X)-XTEN-dCas9; X=D, G or V:

(SEQ ID NO: 28) MSEVEFS Y EYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFR MRRQ X IKAQKKAQSSTD SGSETPGTSESATPES DKKYSIGLAIGTNSVGW AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD ABE7.7: ecTadA_((wild type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 691) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-624 amino acid sequence: ecTadA_((wild type))-32 a.a. linker-ecTadA_((w23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-24 a.a. linker_nCas9_SGGS_NLS

(SEQ ID NO: 692) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPES DKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIFIDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDSGGS PKKKRK V ABE3.2: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((L84F_A106V_D108N_H123Y_D147Y_E155V_I156F))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 693) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMR RQVFKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV ABE5.3: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 694) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-558 amino acid sequence: ecTadA_((wild-type))-32 a.a. linker-ecTadA_((H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N))-24 a.a. linker_nCas9_SGGS_NLS

(SEQ ID NO: 695) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS S EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMR RQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPES DKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGDSGGS PKKKRKV pNMG-576 amino acid sequence: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 696) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-577 amino acid sequence: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 697) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-586 amino acid sequence: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 698) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV ABE7.2: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 699) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-620 amino acid sequence: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 700) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENOTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLONEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-617 amino acid sequence: ecTadA(wild-type)-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 701) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-618 amino acid sequence: ecTadA_((wild-type))-(SGGS)2-XTEN-(SGGS)2-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N))-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

(SEQ ID NO: 702) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMP RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-620 amino acid sequence: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_GGS_NLS

(SEQ ID NO: 703) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGS PKKKRKV pNMG-621 amino acid sequence: ecTadA_((wild-type))-32 a.a. linker-ecTadA_((H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-24 a.a. linker_nCas9_GGS_NLS

(SEQ ID NO: 704) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS S EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPES DKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGDSGGS PKKKRKV pNMG-622 amino acid sequence: ecTadA_((wild-type))-32 a.a. linker-ecTadA_((H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N))-24 a. a. linker_nCas9_GGS_NLS

(SEQ ID NO: 705) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS S EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMP RQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPES DKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGDSGGS PKKKRKV pNMG-623 amino acid sequence: ecTadA_((wild-type))-32 a.a. linker-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-24 a. a. linker_nCas9_GGS_NLS

(SEQ ID NO: 706) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS S EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPES DKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGDSGGS PKKKRKV ABE6.3: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 707) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSPKKKRKV ABE6.4: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F m57N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 708) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSPKKKRKV ABE7.8: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 709) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMR RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSPKKKRKV ABE7.9: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS))-₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 710) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRGEGWNRAIGLHDP TAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVF GVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMPRQ VFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYS IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSGGSPKKKRKV ABE7.10: ecTadA_((wild-type))-(SGGS)₂-XTEN-(SGGS)₂-ecTadA_((w23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N))-(SGGS)₂-XTEN-(SGGS)₂_nCas9_SGGS_NLS

(SEQ ID NO: 711) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSPKKKRKV

In some embodiments, the fusion protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 11-28, 387, 388, 440, 691-711, or to any of the fusion proteins provided herein. In some embodiments, the fusion protein comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 11-28, 387, 388, 440, 691-711, or any of the fusion proteins provided herein. In some embodiments, the fusion protein comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1750, or at least 1800 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 11-28, 387, 388, 440, 691-711, or any of the fusion proteins provided herein.

Complexes of Nucleic Acid Programmable DNA Binding Proteins (napDNAbp) with Guide Nucleic Acids

Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, for example any of the adenosine base editors provided herein, and a guide nucleic acid bound to napDNAbp of the fusion protein. In some embodiments, the disclosure provides any of the fusion proteins (e.g., adenosine base editors) provided herein bound to any of the guide RNAs provided herein. In some embodiments, the napDNAbp of the fusion protein (e.g., adenosine base editor) is a Cas9 domain (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase), which is bound to a guide RNA. In some embodiments, the complexes provided herein are configured to generate a mutation in a nucleic acid, for example to correct a point mutation in a gene (e.g., HFE, HBB, or F8), or to mutate a promoter (e.g., an HBG1 or HBG2 promoter) to modulate expression of one or more proteins (e.g., gamma globin protein) that are under control of the promoter.

In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a target sequence, for example a target DNA sequence. In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a DNA sequence in a promoter region of an HBG1 or HBG2 gene, for example a promoter region of a human HBG1 or HBG2 gene. In some embodiments, the human hemoglobin subunit gamma 1 (HBG1) is the HBG1 of Gene ID: 3047. In some embodiments, the human hemoglobin subunit gamma 2 (HBG2) is the HBG2 of Gene ID: 3048. In some embodiments, the HBG1 or HBG2 promoter is a human, chimpanzee, ape, monkey, dog, mouse, or rat promoter. In some embodiments, the HBG1 or HBG2 promoter is a human HBG1 or HBG2 promoter. In some embodiments, the HBG1 or HBG2 promoter is from 100 to 300 nucleotides upstream of an HBG1 or HBG2 gene. In some embodiments, the HBG1 or HBG2 promoter is from 100 to 300, 110 to 290, 120 to 280, 130 to 270, 140 to 260, 150 to 250, 160 to 240, 160 to 230, 170 to 220, 180 to 210 or from 190 to 200 nucleotides upstream of an HBG1 or HBG2 gene. In some embodiments, the promoter that drives HBG1 expression comprises a T that is 198 nucleotides upstream of HBG1 (−198T). In some embodiments, the gRNA complexed with any of the fusion proteins provided herein (e.g. adenosine base editors) is designed to target the T at position −198 relative to the HBG1 or HBG2, leading to the mutation of the T to a C. Exemplary HBG1 and HBG2 promoter sequences are provided as SEQ ID NO: 344 and 345, respectively. It should be appreciated that the exemplary HBG1 and HBG2 promoter regions are exemplary and are not meant to be limiting.

In some embodiments, the HBG1 or HBG2 promoter comprises the nucleic acid sequence of any one of the below sequences, such as SEQ ID NOs 838-846, 297-323 and SEQ ID NOs 838-846 having a CCT at the 5′ end of the nucleic acid. In some embodiments, the T that is targeted for mutation to a C is indicated in bold in the below sequences.

(SEQ ID NO: 838) 5′-CTTGGGGGCCCCTTCCCCACACTA-3′; (SEQ ID NO: 839) 5′-CTTGGGGGCCCCTTCCCCACACT-3′; (SEQ ID NO: 840) 5′-CTTGGGGGCCCCTTCCCCACAC-3′; (SEQ ID NO: 841) 5′-CTTGGGGGCCCCTTCCCCACA-3′; (SEQ ID NO: 842) 5′-CTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 843) 5′-CTTGGGGGCCCCTTCCCCA-3′; (SEQ ID NO: 844) 5′-CTTGGGGGCCCCTTCCCC-3′; (SEQ ID NO: 845) 5′-CTTGGGGGCCCCTTCCC-3′; (SEQ ID NO: 6) 5′-CCTCTTGGGGGCCCCTTCCCCACACTA-3′; (SEQ ID NO: 7) 5′-CCTCTTGGGGGCCCCTTCCCCACACT-3′; (SEQ ID NO: 15) 5′-CCTCTTGGGGGCCCCTTCCCCACAC-3′; (SEQ ID NO: 16) 5′-CCTCTTGGGGGCCCCTTCCCCACA-3′; (SEQ ID NO: 17) 5′-CCTCTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 18) 5′-CCTCTTGGGGGCCCCTTCCCCA-3′; (SEQ ID NO: 19) 5′-CCTCTTGGGGGCCCCTTCCCC-3′; or (SEQ ID NO: 20) 5′-CCTCTTGGGGGCCCCTTCCC-3′.

In some embodiments, any of the complexes provided herein comprise a gRNA having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to any one of the nucleic acid sequences provided above (e.g., SEQ ID NOs 838-845, 297-323 and SEQ ID NOs 838-845 further comprising a CCT at the 5′ end). It should be appreciated that the guide sequence of the gRNA may comprise one or more nucleotides that are not complementary to a target sequence. In some embodiments, the guide sequence of the gRNA is at the 5′ end of the gRNA. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. In some embodiments, the G at the 5′ end of the gRNA is not complementary with the target sequence. In some embodiments, the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to a target sequence (e.g., any of the target sequences provided herein). It should be appreciated that promoter sequences may vary between the genomes of individuals. Thus, the disclosure provides gRNAs having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a HBG1 or HBG2 promoter target sequence in the genome of a human.

In some embodiments, the gRNA comprises a guide sequence comprising any one of SEQ ID NOs: 846-853 and 254-280, provided below.

(SEQ ID NO: 846) 5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 847) 5′-CAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 848) 5′-AUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 849) 5′-UGUGGGGAAGGGGCCCCCAAG-3′. (SEQ ID NO: 850) 5′-GUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 851) 5′-UGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 852) 5′-GGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 853) 5′-GGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 254) 5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 255) 5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 256) 5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 257) 5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 258) 5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 259) 5′-AUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 260) 5′-AUGCAAAUAUCUGUCUGAAACGG-3′; (SEQ ID NO: 261) 5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 262) 5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 263) 5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 264) 5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 265) 5′-AUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 266) 5′-UAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 267) 5′-AGAUAUUUGCAUUGAGAUAGUGU-3′; (SEQ ID NO: 268) 5′-ACAGAUAUUUGCAUUGAGAUAGU-3′; (SEQ ID NO: 269) 5′- GUGGGGAAGGGGCCCCCAAGAGG-3′; (SEQ ID NO: 270) 5′-CUUGACCAAUAGCCUUGACAAGG-3′; (SEQ ID NO: 271) 5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 272) 5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 273) 5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 274) 5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 275) 5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 276) 5′-CAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 277) 5′-UUGUCAAGGCUAUUGGUCAAGGC-3′; (SEQ ID NO: 278) 5′-CUUGUCAAGGCUAUUGGUCAAGG-3′; (SEQ ID NO: 279) 5′-UUGACCAAUAGCCUUGACAAGGC-3′; or (SEQ ID NO: 280) 5′-UAGCCUUGACAAGGCAAACUUGA-3′;

Given that target sequences in the genomes of individuals may vary, it should be appreciated that the RNA sequences provided in SEQ ID NOs: 846-853, and 254-280 may vary by one or more nucleobases. In some embodiments, the guide sequence of any of the guide RNA sequences provided herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to any on of SEQ ID NOs: 846-853, and 254-280. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. Accordingly, the application provides SEQ ID NOs: 846-853, and 254-280, further comprising a G at their 5′ ends.

In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a target sequence, for example a target DNA sequence in a hemochromatosis (HFE) gene. In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a DNA sequence in a human HFE gene. In some embodiments, the HFE gene is a human, chimpanzee, ape, monkey, dog, mouse, or rat HFE gene. In some embodiments, the HFE gene is a human HFE gene. In some embodiments, the HFE gene is the HFE gene of Gene ID: 3077, which has also been referred to as HH, HFE1, HLA-H, MVCD7, and TFQTL2. Without wishing to be bound by any particular theory, the HFE protein encoded by this gene is a membrane protein that is similar to MHC class I-type proteins and associates with beta2-microglobulin (beta2M). It is thought that this protein functions to regulate iron absorption by regulating the interaction of the transferrin receptor with transferrin. The iron storage disorder, hereditary haemochromatosis, is a recessive genetic disorder that results from defects in this gene. At least nine alternatively spliced variants have been described for this gene. Additional variants have also been found.

An exemplary coding sequence of the HFE gene is shown below, where the wild-type G that is mutated to an A, causing the Cys (C) to Tyr (Y) mutation at amino acid residue 282 (C282Y), is shown in bold and underlining. In some embodiments, this mutation causes hemochromatosis (e.g., hereditary hemochromatosis):

(SEQ ID NO: 871) ATGGGCCCGCGAGCCAGGCCGGCGCTTCTCCTCCTGATGCTTTTGCAGAC CGCGGTCCTGCAGGGGCGCTTGCTGCGTTCACACTCTCTGCACTACCTCT TCATGGGTGCCTCAGAGCAGGACCTTGGTCTTTCCTTGTTTGAAGCTTTG GGCTACGTGGATGACCAGCTGTTCGTGTTCTATGATCATGAGAGTCGCCG TGTGGAGCCCCGAACTCCATGGGTTTCCAGTAGAATTTCAAGCCAGATGT GGCTGCAGCTGAGTCAGAGTCTGAAAGGGTGGGATCACATGTTCACTGTT GACTTCTGGACTATTATGGAAAATCACAACCACAGCAAGGAGTCCCACAC CCTGCAGGTCATCCTGGGCTGTGAAATGCAAGAAGACAACAGTACCGAGG GCTACTGGAAGTACGGGTATGATGGGCAGGACCACCTTGAATTCTGCCCT GACACACTGGATTGGAGAGCAGCAGAACCCAGGGCCTGGCCCACCAAGCT GGAGTGGGAAAGGCACAAGATTCGGGCCAGGCAGAACAGGGCCTACCTGG AGAGGGACTGCCCTGCACAGCTGCAGCAGTTGCTGGAGCTGGGGAGAGGT GTTTTGGACCAACAAGTGCCTCCTTTGGTGAAGGTGACACATCATGTGAC CTCTTCAGTGACCACTCTACGGTGTCGGGCCTTGAACTACTACCCCCAGA ACATCACCATGAAGTGGCTGAAGGATAAGCAGCCAATGGATGCCAAGGAG TTCGAACCTAAAGACGTATTGCCCAATGGGGATGGGACCTACCAGGGCTG GATAACCTTGGCTGTACCCCCTGGGGAAGAGCAGAGATATACGT G CCAGG TGGAGCACCCAGGCCTGGATCAGCCCCTCATTGTGATCTGGGAGCCCTCA CCGTCTGGCACCCTAGTCATTGGAGTCATCAGTGGAATTGCTGTTTTTGT CGTCATCTTGTTCATTGGAATTTTGTTCATAATATTAAGGAAGAGGCAGG GTTCAAGAGGAGCCATGGGGCACTACGTCTTAGCTGAACGTGAGTGA

An exemplary HFE amino acid sequence, encoded from the above HFE nucleic acid coding sequence, is shown below in (SEQ ID NO: 750), where the C at position 282, which is mutated to a Y in hemochromatosis, is indicated in bold and underlining:

(SEQ ID NO: 750) MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEAL GYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTV DFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCP DTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRG VLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKE FEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPS PSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAER.

In some embodiments, the HFE gene comprises a G to A mutation in the coding sequence of the HFE gene, which causes C to Y mutation in the HFE protein. For example a C282Y mutation in SEQ ID NO: 750. In some embodiments, the HFE gene comprises a G to A mutation in nucleic acid residue 845 of SEQ ID NO: 871, which causes C to Y mutation in the encoded HFE protein. In some embodiments, complexes provided herein are designed to correct the C to Y mutation in HFE (e.g., a C282Y mutation of SEQ ID NO: 750) that causes hemochromatosis. It should be appreciated that the coding sequence of HFE may vary between individuals. Thus, the guide sequence of any of the gRNAs provided herein may be engineered to account for such differences to correct the C to Y mutation in HFE that causes hemochromatosis.

In some embodiments, the HFE gene comprises the nucleic acid sequence of any one of the below sequences, such as SEQ ID NOs 854-861 and SEQ ID NOs 854-861 having a CCT at the 5′ end of the nucleic acid. In some embodiments, the T that is targeted for mutation to a C is indicated in bold in the below sequences. The A opposite of the targeted T may be deaminated using any of the complexes provided herein. The sequences provided below are reverse complements of portions of the coding sequence of an HFE gene.

(SEQ ID NO: 854) 5′-GGGTGCTCCACCTGGTACGTATAT-3′; (SEQ ID NO: 855) 5′-GGGTGCTCCACCTGGTACGTATA-3′; (SEQ ID NO: 856) 5′-GGGTGCTCCACCTGGTACGTAT-3′; (SEQ ID NO: 857) 5′-GGGTGCTCCACCTGGTACGTA-3′; (SEQ ID NO: 858) 5′-GGGTGCTCCACCTGGTACGT-3′; (SEQ ID NO: 859) 5′-GGGTGCTCCACCTGGTACG-3′; (SEQ ID NO: 860) 5′-GGGTGCTCCACCTGGTAC-3′; (SEQ ID NO: 861) 5′-GGGTGCTCCACCTGGTA-3′; (SEQ ID NO: 21) 5′-CCTGGGTGCTCCACCTGGTACGTATAT-3′; (SEQ ID NO: 22) 5′-CCTGGGTGCTCCACCTGGTACGTATA-3′; (SEQ ID NO: 23) 5′-CCTGGGTGCTCCACCTGGTACGTAT-3′; (SEQ ID NO: 24) 5′-CCTGGGTGCTCCACCTGGTACGTA-3′; (SEQ ID NO: 25) 5′-CCTGGGTGCTCCACCTGGTACGT-3′; (SEQ ID NO: 26) 5′-CCTGGGTGCTCCACCTGGTACG-3′; (SEQ ID NO: 29) 5′-CCTGGGTGCTCCACCTGGTAC-3′; or (SEQ ID NO: 30) 5′-CCTGGGTGCTCCACCTGGTA-3′.

In some embodiments, any of the complexes provided herein comprise a gRNA having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to any one of the nucleic acid sequences provided above (e.g., SEQ ID NOs 854-861 and SEQ ID NOs 854-861 having a CCT at the 5′ end). It should be appreciated that the guide sequence of the gRNA may comprise one or more nucleotides that are not complementary to a target sequence. In some embodiments, the guide sequence of the gRNA is at the 5′ end of the gRNA. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. In some embodiments, the G at the 5′ end of the gRNA is not complementary with the target sequence. In some embodiments, the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to a target sequence (e.g., any of the target sequences provided herein). It should be appreciated that HFE gene sequences may vary between the genomes of individuals. Thus, the disclosure provides gRNAs having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a HFE gene target sequence in the genome of a human.

In some embodiments, the gRNA comprises a guide sequence comprising any one of SEQ ID NOs: 862-869, provided below.

(SEQ ID NO: 862) 5′-AUAUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 863) 5′-UAUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 864) 5′-AUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 865) 5′-UACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 866) 5′-ACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 867) 5′-CGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 868) 5′-GUACCAGGUGGAGCACCC-3′; or (SEQ ID NO: 869) 5′-UACCAGGUGGAGCACCC-3′.

Given that target sequences in the genomes of individuals may vary, it should be appreciated that the RNA sequences provided in SEQ ID NOs: 862-869 may vary by one or more nucleobases. In some embodiments, the guide sequence of any of the guide RNA sequences provided herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to any of SEQ ID NOs: 862-869. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. Accordingly, the application provides SEQ ID NOs: 862-869 that further comprise a G at their 5′ ends.

In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a target sequence, for example a target DNA sequence in a beta globin (HBB) gene. In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a DNA sequence in a human HBB gene. In some embodiments, the HBB gene is a human, chimpanzee, ape, monkey, dog, mouse, or rat HBB gene. In some embodiments, the HBB gene is a human HBB gene. In some embodiments, the HBB gene is the HBB gene of Gene ID: 3043, which has also been referred to as ECYT6, CD113t-C, and beta-globin. Without wishing to be bound by any particular theory, the hemoglobin subunit beta is a globin protein, which along with alpha globin (HBA), makes up the most common form of haemoglobin in adult humans. Mutations in the gene produce several variants of the proteins which are implicated with genetic disorders such as sickle-cell disease and beta thalassemia, including hemoglobin C disease (Hb C) and hemoglobin E disease (Hb E).

An exemplary coding sequence of the HBB gene is shown below. In some embodiments, this sequence is mutated (e.g., A to T mutation) to cause sickle cell disease (E6V mutation in the protein). In some embodiments, this sequence is mutated (e.g., G to A mutation) to cause Hb C (E6K mutation in the protein). In some embodiments, this sequence is mutated (e.g., G to A mutation) to cause HbE (E26K mutation in the protein).

Exemplary HBB Gene:

(SEQ ID NO: 346) ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG CAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTAT CAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGA GACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTAT TGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAG AGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG CAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTG ATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGT GAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAG TCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTT CATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAAC AGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTT TTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCT TTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATT GTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAA AAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGC TTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATT GATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATA TGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAA AAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATA CTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGC CTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCA ATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGAT GTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTG CTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAG GCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCT GGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCA CCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAAT GCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCT ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATT ATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTT CATTGC

An exemplary HBB amino acid sequence, is shown below in (SEQ ID NO: 340), where the E at position 6, which is mutated to a V in sickle cell disease or to a K in Hb C, is indicated in bold and underlining. The E at position 26, which is mutated to a K in Hb E is also indicated in bold and underlining.

(SEQ ID NO: 340) VHLTP E EKSAVTALWGKVNVDEVGG E ALGRLLVVYPWTQRFFESFGDLST PDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDP ENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

In some embodiments, the HBB gene comprises a G to A or A to T mutation in the coding sequence of the HBB gene, which causes a E6V, a E6K, or a E26K mutation in HBB. For example, a E6V, a E6K, or a E26K mutation in SEQ ID NO: 340. In some embodiments, complexes provided herein are designed to correct the E6V (e.g., changing pathogenic V mutation to non-pathogenic A mutation), a E6K, or a E26K mutation in HBB, that causes sickle cell disease, Hb C, and Hb E, respectively. It should be appreciated that the coding sequence of HBB may vary between individuals. Thus, the guide sequence of any of the gRNAs provided herein may be engineered to account for such differences to correct the mutations provided herein.

In some embodiments, the HBB gene comprises the nucleic acid sequence, or a reverse complement thereof, of any one of the below sequences, such as SEQ ID NOs 324-337. In some embodiments, the T that is targeted for mutation to a C is indicated in bold in the below sequences. The A opposite of the targeted T may be deaminated using any of the complexes provided herein.

(SEQ ID NO: 324) 5′-GTGCATCTGACTCCTGTGGAGAA-3′; (SEQ ID NO: 325) 5′-GGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 326) 5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′; (SEQ ID NO: 327) 5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 328) 5′-CCATGGTGCATCTGACTCCTGTGGAG-3′; (SEQ ID NO: 329) 5′-CCATGGTGCATCTGACTCCTGTGGA-3′; (SEQ ID NO: 330) 5′-CCATGGTGCATCTGACTCCTGTGG-3′; (SEQ ID NO: 331) 5′-CCATGGTGCATCTGACTCCTGTG-3′; (SEQ ID NO: 332) 5′-GCATCTGACTCCTGTGGAGAAGT-3′; (SEQ ID NO: 333) 5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 334) 5′-ACGGCAGACTTCTCCTTAGGAGT-3′; (SEQ ID NO: 335) 5′-CCTGCCCAGGGCCTTACCACCAA-3′; (SEQ ID NO: 336) 5′-ACCTGCCCAGGGCCTTACCACCA-3′; or (SEQ ID NO: 337) 5′-CCAACCTGCCCAGGGCCTTACCA-3′

In some embodiments, any of the complexes provided herein comprise a gRNA having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to any one of the nucleic acid sequences provided above (e.g., SEQ ID NOs 324-337). It should be appreciated that the guide sequence of the gRNA may comprise one or more nucleotides that are not complementary to a target sequence. In some embodiments, the guide sequence of the gRNA is at the 5′ end of the gRNA. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. In some embodiments, the G at the 5′ end of the gRNA is not complementary with the target sequence. In some embodiments, the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to a target sequence (e.g., any of the target sequences provided herein). It should be appreciated that BB gene sequences may vary between the genomes of individuals. Thus, the disclosure provides gRNAs having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a HBB gene target sequence in the genome of a human.

In some embodiments, the gRNA comprises a guide sequence comprising any one of SEQ ID NOs: 281-294, provided below. SEQ ID NOs: 281-290 are designed to treat sickle cell disease (e.g., changing E6V mutation to have an A at position 6, which is non-pathogenic). SEQ ID NO: 291 is designed to treat Hb C (e.g., E6K mutation). SEQ ID NOs: 292-294 are designed to Hb E (e.g., E26K mutation).

(SEQ ID NO: 281) 5′-UUCUCCACAGGAGUCAGAUGCAC-3′; (SEQ ID NO: 282) 5′-UCUCCACAGGAGUCAGAUGCACC-3′; (SEQ ID NO: 283) 5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 284) 5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 285) 5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 286) 5′-UCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 287) 5′-CCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 288) 5′-CACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 289) 5′-ACUUCUCCACAGGAGUCAGAUGC-3′; (SEQ ID NO: 290) 5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′; (SEQ ID NO: 291) 5′-ACUCCUAAGGAGAAGUCUGCCGU-3′; (SEQ ID NO: 292) 5′-UUGGUGGUAAGGCCCUGGGCAGG-3′; (SEQ ID NO: 293) 5′-UGGUGGUAAGGCCCUGGGCAGGU-3′; or (SEQ ID NO: 294) 5′-UGGUAAGGCCCUGGGCAGGUUGG-3′.

Given that target sequences in the genomes of individuals may vary, it should be appreciated that the RNA sequences provided in SEQ ID NOs: 281-294 may vary by one or more nucleobases. In some embodiments, the guide sequence of any of the guide RNA sequences provided herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to any of SEQ ID NOs: 281-294. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. Accordingly, the application provides SEQ ID NOs: 281-294 that further comprise a G at their 5′ ends.

In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a target sequence, for example a target DNA sequence in a coagulation factor VIII (F8) gene. In some embodiments, the guide RNA comprises a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a DNA sequence in a human F8 gene. In some embodiments, the F8 gene is a human, chimpanzee, ape, monkey, dog, mouse, or rat F8 gene. In some embodiments, the F8 gene is a human F8 gene. In some embodiments, the F8 gene is the F8 gene of Gene ID: 2157, which has also been referred to as AHF, F8B, F8C, HEMA, FVIII, and DXS1253E. Without wishing to be bound by any particular theory, F8 encodes coagulation factor VIII, which participates in the intrinsic pathway of blood coagulation; factor VIII is a cofactor for factor IXa which, in the presence of Ca⁺² and phospholipids, converts factor X to the activated form Xa. Defects in this gene results in hemophilia A, a common recessive X-linked coagulation disorder.

An exemplary coding sequence of the F8 gene is provided as SEQ ID NO: 347. In some embodiments, this sequence is mutated (e.g., C to T mutation) to cause hemophilia A (R612C mutation in the protein).

An exemplary coagulation factor VIII amino acid sequence, is shown below in (SEQ ID NO: 341), where the R at position 612, which is mutated to a C in hemophilia A is indicated in bold and underlining.

(SEQ ID NO: 341) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFP PRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVY DTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPG GSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRE GSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNH RQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPE EPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTR YYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE NRSWYLTENIQ R FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCL HEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMS MENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLL SKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPK IQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSL SEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTE SGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQN ILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKK EGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVS LGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDN LHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLS TRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLG NQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRI IVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIP QANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQES SHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLP KPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEG AIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWK SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGR TERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYD EDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFK KVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASR PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFD CKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGH IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDL Y

In some embodiments, the F8 gene comprises a C to T mutation in the coding sequence of the F8 gene, which causes an R to C mutation in coagulation factor VIII. For example, an R612C mutation in SEQ ID NO: 341. In some embodiments, complexes provided herein are designed to correct the R to C (e.g., R612C) mutation in F8, which causes hemophilia A. It should be appreciated that the coding sequence of F8 may vary between individuals. Thus, the guide sequence of any of the gRNAs provided herein may be engineered to account for such differences to correct the mutations provided herein.

In some embodiments, the F8 gene comprises the nucleic acid sequence, or a reverse complement thereof, of any one of the below sequences, such as SEQ ID NOs 338-339. In some embodiments, the T that is targeted for mutation to a C is indicated in bold in the below sequences. The A opposite of the targeted T may be deaminated using any of the complexes provided herein.

(SEQ ID NO: 338) 5′-CCTCACAGAGAATATACAATGCT-3′; or (SEQ ID NO: 339) 5′-TCACAGAGAATATACAATGCTTT-3′.

In some embodiments, any of the complexes provided herein comprise a gRNA having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to any one of the nucleic acid sequences provided above (e.g., SEQ ID NOs 338-339). It should be appreciated that the guide sequence of the gRNA may comprise one or more nucleotides that are not complementary to a target sequence. In some embodiments, the guide sequence of the gRNA is at the 5′ end of the gRNA. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. In some embodiments, the G at the 5′ end of the gRNA is not complementary with the target sequence. In some embodiments, the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to a target sequence (e.g., any of the target sequences provided herein). It should be appreciated that F8 gene sequences may vary between the genomes of individuals. Thus, the disclosure provides gRNAs having a guide sequence that comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleic acids that are 100% complementary to a F8 gene target sequence in the genome of a human.

In some embodiments, the gRNA comprises a guide sequence comprising any one of SEQ ID NOs: 295-296, provided below, which are designed treat hemophilia A (e.g., R612C mutation, e.g., of SEQ ID NO: 341).

(SEQ ID NO: 295) 5′-AGCAUUGUAUAUUCUCUGUGAGG-3′; or (SEQ ID NO: 296) 5′-AAAGCAUUGUAUAUUCUCUGUGA-3′

Given that target sequences in the genomes of individuals may vary, it should be appreciated that the RNA sequences provided in SEQ ID NOs: 295-296 may vary by one or more nucleobases. In some embodiments, the guide sequence of any of the guide RNA sequences provided herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to any of SEQ ID NOs: 295-295. In some embodiments, the guide sequence of the gRNA further comprises a G at the 5′ end of the gRNA. Accordingly, the application provides SEQ ID NOs: 295-296 that further comprise a G at their 5′ ends.

In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-150 nucleotides long and comprises a guide sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides long. In some embodiments, the guide RNA comprises a guide sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence, for example any of the HBG1 or HBG2 promoter sequences provided herein or any of the HFE, HBB, or F8 target sequences provided herein. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with a disease or disorder, e.g., hereditary persistence of fetal hemoglobin (HPFH), beta-thalassemia, hereditary hemochromatosis (HHC), sickle cell disease, Hb C, Hb E, or hemophilia (e.g., hemophilia A).

Methods of Using Fusion Proteins Comprising an Adenosine Deaminase and a Nucleic Acid Programmable DNA Binding Protein (napDNAbp) Domain

Some aspects of this disclosure provide methods of using the fusion proteins, or complexes comprising a guide nucleic acid (e.g., gRNA) and a nucleobase editor provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA, or RNA molecule with any of the fusion proteins provided herein, and with at least one guide nucleic acid (e.g., guide RNA), wherein the guide nucleic acid, (e.g., guide RNA) is comprises a sequence (e.g., a guide sequence that binds to a DNA target sequence) of at least 10 (e.g., at least 10, 15, 20, 25, or 30) contiguous nucleotides that is 100% complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

Expressing Hemoglobin

Some aspects of the disclosure provide methods of introducing disease-suppressing mutations in cells (e.g., mammalian cells). In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to modulate expression of a hemoglobin gene (e.g., HBG1 and/or HBG2). In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to T and/or T to C mutation in a promoter region of HBG1 and/or HBG2.

Humans with the rare benign condition hereditary persistence of fetal hemoglobin (HPFH) are resistant to some β-globin diseases including sickle-cell anemia. In certain patients, this phenotype is mediated by mutations in the promoters of the γ-globin genes HBG1 and HBG2 that enable sustained expression of fetal hemoglobin, which is normally silenced in humans around birth. Without wishing to be bound by any particular theory, generating one or more mutations in a promoter region of HBG1 or HBG2 can increase expression of HBG1 and/or HBG2 in order to treat β-globin diseases.

In some embodiments, the methods include deaminating an adenosine nucleobase (A) in a promoter of an HBG1 or HBG2 gene by contacting the promoter with a base editor and a guide RNA bound to the base editor, where the guide RNA (gRNA) comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter of the HBG1 and/or HBG2 gene. It should be appreciated that the prompter of the HBG1 and HBG2 genes can include any of the sequences of the HBG1 and HBG2 promoters described herein. In some embodiments, the guide sequence of the gRNA comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence in a promoter sequence of HBG1 or HBG2.

In some embodiments, the methods further comprising nicking the target sequence, which may be achieved by using a nucleic acid programmable binding protein (napDNAbp), such as a Cas9 nickase (nCas9) that nicks the target sequence that is complementary to the guide sequence of the gRNA. In some embodiments, the target nucleic acid sequence in the promotor comprises the nucleic acid sequence

(SEQ ID NO: 838) 5′-CTTGGGGGCCCCTTCCCCACACTA-3′; (SEQ ID NO: 839) 5′-CTTGGGGGCCCCTTCCCCACACT-3′; (SEQ ID NO: 840) 5′-CTTGGGGGCCCCTTCCCCACAC-3′; (SEQ ID NO: 841) 5′-CTTGGGGGCCCCTTCCCCACA-3′; (SEQ ID NO: 842) 5′-CTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 843) 5′-CTTGGGGGCCCCTTCCCCA-3′; (SEQ ID NO: 844) 5′-CTTGGGGGCCCCTTCCCC-3′; (SEQ ID NO: 845) 5′-CTTGGGGGCCCCTTCCC-3′; or any one of SEQ ID NOs: 297-323. It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 838-845 may further comprise 5′-CCT-3′ at their 5′ end.

In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence

(SEQ ID NO: 846) 5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 847) 5′-CAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 848) 5′-AUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 849) 5′-UGUGGGGAAGGGGCCCCCAAG-3′. (SEQ ID NO: 850) 5′-GUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 851) 5′-UGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 852) 5′-GGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 853) 5′-GGGAAGGGGCCCCCAAG-3′ or any one of SEQ ID NOs: 254-280.

It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 846-853, or 254-280 may further comprise a G at their 5′ end.

In some embodiments, deaminating the adenosine nucleobase in the promoter of HBG1 or HBG2 results in a T-A base pair in the promoter being mutated to a C-G base pair in the promoter. In some embodiments, deaminating the adenosine nucleobase in the promoter results in a sequence associated with hereditary persistence of fetal hemoglobin (HPFH). Hereditary persistence of fetal hemoglobin, in some embodiments, is characterized as a benign condition in which fetal hemoglobin (e.g., hemoglobin F) is expressed in adulthood. In some embodiments, HPFH is characterized by expression of fetal hemoglobin in a subject of 2 years or older, 5 years or older, 10 years or older, 15 years or older 20 years or older, 25 years or older, or 30 years or older. In some embodiments, HPFH is considered to be expressed in an adult if it is expressed at a level that is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more greater than in a normal subject of 2 years or older, 5 years or older, 10 years or older, 15 years or older 20 years or older, 25 years or older, or 30 years or older.

In some embodiments, deaminating the adenosine nucleobase in the promoter of the HBG1 gene leads to an increase in transcription of the HBG1 gene by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to a healthy adult or a fetus that expresses HBG1. In some embodiments, deaminating the adenosine nucleobase in the promoter of the HBG2 gene leads to an increase in transcription of the HBG2 gene by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to a healthy adult or a fetus that expresses HBG1. In some embodiments, deaminating the adenosine nucleobase in the promoter of the HBG1 gene leads to an increase HBG1 protein levels by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to a healthy adult or a fetus that expresses HBG1. In some embodiments, deaminating the adenosine nucleobase in the promoter of the HBG2 gene leads to an increase in HBG2 protein levels by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to a healthy adult or a fetus that expresses HBG2.

In some embodiments, the method is performed in vitro, for example in cultured cells. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in the cell of a subject. In some embodiments, the subject has or is suspected of having a disease or disorder of the blood. In some embodiments, the disease or disorder is an anemia. In some embodiments, the anemia is iron deficiency anemia, aplastic anemia, haemolytic anemia, thalassaemia, sickle cell anemia, pernicious anemia, or fanconi anemia. In some embodiments, the disease or disorder is caused by a mutation in a gene or a promoter of a gene encoding a globin protein, for example CYGB, HBA1, HBA2, HBB, HBD, HBE1, HBG1, HBG2, HBM, HBQ1, HBZ, or MB.

Correcting Mutations in an HFE Gene

Some aspects of the disclosure provide methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to correct a point mutation in an HFE gene. In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to G and/or T to C mutation in an HFE gene. In some embodiments, the disclosure provides method for deaminating an adenosine nucleobase (A) in an HFE gene, the method comprising contacting the HFE gene with a base editor and a guide RNA bound to the base editor, where the guide RNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the HFE gene. In some embodiments, the HFE gene comprises a C to T or G to A mutation. In some embodiments, the C to T or G to A mutation in the HFE gene impairs function of the HFE protein encoded by the HFE gene. In some embodiments, the C to T or G to A mutation in the HFE gene impairs function of the HFE protein encoded by the HFE gene by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%. In some embodiments, the function of the HFE protein is iron absorption.

In some embodiments, deaminating an adenosine (A) nucleobase complementary to the T corrects the C to T or G to A mutation in the HFE gene. In some embodiments, the C to T or G to A mutation in the HFE gene leads to a Cys (C) to Tyr (Y) mutation in the HFE protein encoded by the HFE gene. In some embodiments, deaminating the adenosine nucleobase complementary to the T corrects the Cys to Tyr mutation in the HFE protein.

In some embodiments, the guide sequence of the gRNA comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence of the HFE gene. In some embodiments, the base editor nicks the target sequence that is complementary to the guide sequence. In some embodiments, the target nucleic acid sequence in the HFE gene comprises the nucleic acid sequence:

(SEQ ID NO: 854) 5′-GGGTGCTCCACCTGGTACGTATAT-3′; (SEQ ID NO: 855) 5′-GGGTGCTCCACCTGGTACGTATA-3′; (SEQ ID NO: 856) 5′-GGGTGCTCCACCTGGTACGTAT-3′; (SEQ ID NO: 857) 5′-GGGTGCTCCACCTGGTACGTA-3′; (SEQ ID NO: 858) 5′-GGGTGCTCCACCTGGTACGT-3′; (SEQ ID NO: 859) 5′-GGGTGCTCCACCTGGTACG-3′; (SEQ ID NO: 860) 5′-GGGTGCTCCACCTGGTAC-3′; or (SEQ ID NO: 861) 5′-GGGTGCTCCACCTGGTA-3′. It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 854-861 may further comprise 5′-CCT-3′ at their 5′ end.

In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence:

(SEQ ID NO: 862) 5′-AUAUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 863) 5′-UAUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 864) 5′-AUACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 865) 5′-UACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 866) 5′-ACGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 867) 5′-CGUACCAGGUGGAGCACCC-3′; (SEQ ID NO: 868) 5′-GUACCAGGUGGAGCACCC-3′; or (SEQ ID NO: 869) 5′-UACCAGGUGGAGCACCC-3′. It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 862-869 may further comprise a G at their 5′ end.

In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in a T-A base pair in the HFE gene being mutated to a C-G base pair in the HFE gene. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in correcting a sequence associated with hereditary hemochromatosis (HHC). In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in an increase in HFE protein function. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in increase in HFE protein function to at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least 100% as compared to wild-type HFE protein function. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in a decrease in one or more symptoms of hemochromatosis. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in an increase in iron absorption function. In some embodiments, deaminating the adenosine nucleobase in the HFE gene results in an increase in iron absorption function to least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% of a normal level of iron absorption, for example a level of iron absorption in a subject that does not have hemochromatosis. In some embodiments, deaminating the adenosine nucleobase in the HFE gene leads to an increase in function of HFE protein transcribed from the HFE gene. In some embodiments, deaminating the adenosine nucleobase in the HFE gene leads to an increase in HFE stability or half life, for example by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.

In some embodiments, the HFE gene is in a cell, such as a cell in culture (e.g., an immortalized lymphoblastoid cell (LCL)) or a cell in a subject. In some embodiments, the HFE gene encodes an HFE protein comprising a Cys to Tyr mutation. In some embodiments, the HFE protein comprises a Cys to Tyr mutation (C282Y) at residue 282 of the amino acid sequence SEQ ID NO: 750, where the Cys at position 282 is shown in bold:

(SEQ ID NO: 750) MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEAL GYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTV DFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCP DTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRG VLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKE FEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPS PSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAER.

In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in a cell of a subject. In some embodiments, the subject has or is suspected of having an iron storage disorder. In some embodiments, the subject has or is suspected of having hemochromatosis. In some embodiments, the subject has or is suspected of having hereditary hemochromatosis (HHC). In some embodiments, the subject has a mutation in an HFE gene, where a wild-type G that is mutated to an A (e.g., a G845A mutation in SEQ ID NO: 871), causes a Cys (C) to Tyr (Y) mutation, for example at amino acid residue 282 (C282Y) of SEQ ID NO: 750. In some embodiments, this mutation causes hemochromatosis (e.g., hereditary hemochromatosis). In some embodiments, deaminating the adenosine nucleobase in the HFE gene ameliorates one or more symptoms of the iron storage disorder in the subject.

Correcting/Generating Mutations in an HBB Gene

Some aspects of the disclosure provide methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to correct a point mutation in an HBB gene, or generate a point mutation that is non-pathogenic. In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to G and/or T to C mutation in an HBB gene. In some embodiments, the disclosure provides method for deaminating an adenosine nucleobase (A) in an HBB gene, the method comprising contacting the HBB gene with a base editor and a guide RNA bound to the base editor, where the guide RNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the HBB gene. In some embodiments, the HBB gene comprises a A to T or G to A mutation. In some embodiments, the A to T or G to A mutation in the HBB gene impairs function of the beta globin protein encoded by the HBB gene. In some embodiments, the A to T or G to A mutation in the HBB gene impairs function of the HBB protein encoded by the HBB gene by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%. In some embodiments, the function of the HBB protein is oxygen carrying capacity. In some embodiments, the A to T mutation causes sickle cell disease and changing the T:A base pair to a C:G base pair yields a non-pathogenic point mutation that decreases the ability of the hemoglobin to polymerize, e.g., by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.

In some embodiments, deaminating an adenosine (A) nucleobase complementary to the T corrects the C to T or G to A mutation in the HBB gene or generates a non-pathogenic mutation. In some embodiments, the A to T or G to A mutation in the HBB gene leads to an E to V mutation or an E to K mutation in the HBB protein encoded by the HBB gene. In some embodiments, deaminating the adenosine nucleobase complementary to the T corrects the E to K mutation in the HBB protein. For example deaminating the adenosine nucleobase complementary to the T corrects an E6K (Hb C) or an E26K (Hb E) mutation in HBB, for example as compared to SEQ ID NO: 340. In some embodiments, deaminating the adenosine nucleobase complementary to the T changes a pathogenic mutation to a non-pathogenic mutation. For example an E6V mutation in HBB (e.g., as compared to SEQ ID NO: 340) can be mutated to generate an A at position 6 (e.g., V6A mutation) to yield a non-pathogenic mutation.

In some embodiments, the guide sequence of the gRNA comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence of the HBB gene. In some embodiments, the base editor nicks the target sequence that is complementary to the guide sequence. In some embodiments, the target nucleic acid sequence in the HBB gene comprises the nucleic acid sequence:

(SEQ ID NO: 324) 5′-GTGCATCTGACTCCTGTGGAGAA-3′; (SEQ ID NO: 325) 5′-GGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 326) 5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′; (SEQ ID NO: 327) 5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 328) 5′-CCATGGTGCATCTGACTCCTGTGGAG-3′; (SEQ ID NO: 329) 5′-CCATGGTGCATCTGACTCCTGTGGA-3′; (SEQ ID NO: 330) 5′-CCATGGTGCATCTGACTCCTGTGG-3′; (SEQ ID NO: 331) 5′-CCATGGTGCATCTGACTCCTGTG-3′; (SEQ ID NO: 332) 5′-GCATCTGACTCCTGTGGAGAAGT-3′; (SEQ ID NO: 333) 5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′; (SEQ ID NO: 334) 5′-ACGGCAGACTTCTCCTTAGGAGT-3′; (SEQ ID NO: 335) 5′-CCTGCCCAGGGCCTTACCACCAA-3′; (SEQ ID NO: 336) 5′-ACCTGCCCAGGGCCTTACCACCA-3′; or (SEQ ID NO: 337) 5′-CCAACCTGCCCAGGGCCTTACCA-3′.

In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence:

(SEQ ID NO: 281) 5′-UUCUCCACAGGAGUCAGAUGCAC-3′; (SEQ ID NO: 282) 5′-UCUCCACAGGAGUCAGAUGCACC-3′; (SEQ ID NO: 283) 5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 284) 5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 285) 5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 286) 5′-UCCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 287) 5′-CCACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 288) 5′-CACAGGAGUCAGAUGCACCAUGG-3′; (SEQ ID NO: 289) 5′-ACUUCUCCACAGGAGUCAGAUGC-3′; (SEQ ID NO: 290) 5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′; (SEQ ID NO: 291) 5′-ACUCCUAAGGAGAAGUCUGCCGU-3′; (SEQ ID NO: 292) 5′-UUGGUGGUAAGGCCCUGGGCAGG-3′; (SEQ ID NO: 293) 5′-UGGUGGUAAGGCCCUGGGCAGGU-3′; or (SEQ ID NO: 294) 5′-UGGUAAGGCCCUGGGCAGGUUGG-3′. It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 281-294 may further comprise a G at their 5′ end.

In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in a T-A base pair in the HBB gene being mutated to a C-G base pair in the HBB gene. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in correcting a sequence associated with Hb C or Hb E; or results in generating a non-pathogenic mutation. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in an increase in HBB protein function. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in increase in HBB protein function to at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least 100% as compared to wild-type HBB protein function. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in a decrease in one or more symptoms of sickle cell disease, Hb C, or Hb E. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in an increase in oxygen carrying function, or in a decrease in polymerization of beta globin, or a decrease in cell sickling. In some embodiments, deaminating the adenosine nucleobase in the HBB gene results in an increase in oxygen carrying function to least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% of a normal level of oxygen carrying function, for example a level of oxygen carrying function (e.g., oxygen saturation) in a subject that does not have sickle cell, Hb C, and/or Hb E. In some embodiments, deaminating the adenosine nucleobase in the HBB gene leads to an increase in function of HBB protein transcribed from the HBB gene. In some embodiments, deaminating the adenosine nucleobase in the HBB gene leads to an increase in HBB stability or half life, for example by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.

In some embodiments, the HBB gene is in a cell, such as a cell in culture or a cell in a subject. In some embodiments, the HBB gene encodes an HBB protein comprising an E to V or E to K mutation. In some embodiments, the HBB protein comprises a E to V mutation (E6V) at residue 6 of the amino acid sequence SEQ ID NO: 340 (sickle cell disease), where the E at position 6 is shown in bold. In some embodiments, the HBB protein comprises an E to K mutation (E6K) at residue 6 of the amino acid sequence SEQ ID NO: 340 (Hb C), where the E at position 6 is shown in bold. In some embodiments, the HBB protein comprises a E to K mutation (E26K) at residue 6 of the amino acid sequence SEQ ID NO: 340 (Hb E), where the E at position 26 is shown in bold and underlined:

(SEQ ID NO: 340) VHLTPEEKSAVTALWGKVNVDEVGG E ALGRLLVVYPWTQRFFESFGDL STPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKL HVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHK YH.

In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in a cell of a subject. In some embodiments, the subject has or is suspected of having an iron storage disorder. In some embodiments, the subject has or is suspected of having sickle cell disease, or beta-thalassemia. In some embodiments, the subject has or is suspected of having Hb C or Hb E. In some embodiments, the subject has a mutation in an HBB gene, where a wild-type A is mutated to a T, or a wild-type G is mutated to an A. In some embodiments, deaminating the adenosine nucleobase in the HBB gene ameliorates one or more symptoms of sickle cell disease, Hb C, or Hb E in the subject.

Correcting Mutations in an F8 Gene

Some aspects of the disclosure provide methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to correct a point mutation in an F8 gene. In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to G and/or T to C mutation in an F gene. In some embodiments, the disclosure provides method for deaminating an adenosine nucleobase (A) in an F8 gene, the method comprising contacting the F8 gene with a base editor and a guide RNA bound to the base editor, where the guide RNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the F8 gene. In some embodiments, the F8 gene comprises a C to T or G to A mutation. In some embodiments, the C to T or G to A mutation in the F8 gene impairs function of the coagulation factor VIII protein encoded by the F8 gene. In some embodiments, the C to T or G to A mutation in the F8 gene impairs function of the coagulation factor VIII protein encoded by the F8 gene by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%. In some embodiments, the function of the coagulation factor VIII protein is blood clotting.

In some embodiments, deaminating an adenosine (A) nucleobase complementary to the T corrects the C to T or G to A mutation in the F8 gene. In some embodiments, the C to T or G to A mutation in the F8 gene leads to an R to C mutation in the factor VIII protein encoded by the F8 gene. In some embodiments, deaminating the adenosine nucleobase complementary to the T corrects the R to C mutation in the factor VIII protein.

In some embodiments, the guide sequence of the gRNA comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence of the F8 gene. In some embodiments, the base editor nicks the target sequence that is complementary to the guide sequence. In some embodiments, the target nucleic acid sequence in the F8 gene comprises the nucleic acid sequence:

(SEQ ID NO: 338) 5′-CCTCACAGAGAATATACAATGCT-3′; or (SEQ ID NO: 339) 5′-TCACAGAGAATATACAATGCTTT-3′.

In some embodiments, the guide sequence of the gRNA comprises the nucleic acid sequence:

(SEQ ID NO: 295) 5′-AGCAUUGUAUAUUCUCUGUGAGG-3′; or (SEQ ID NO: 296) 5′-AAAGCAUUGUAUAUUCUCUGUGA-3′.

It should be appreciated that any of the the nucleic acids of SEQ ID NOs: 295-296 may further comprise a G at their 5′ end.

In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in a T-A base pair in the F8 gene being mutated to a C-G base pair in the F8 gene. In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in correcting a sequence associated with hemophilia (e.g., hemophilia A). In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in an increase in factor VIII protein function. In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in increase in factor VIII protein function to at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least 100% as compared to wild-type factor VIII protein function. In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in a decrease in one or more symptoms of hemophilia. In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in an increase in blood clotting function. In some embodiments, deaminating the adenosine nucleobase in the F8 gene results in an increase in blood clotting function to least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% of a normal level of blood clotting function, for example a level of blood clotting function in a subject that does not have hemophelia. In some embodiments, deaminating the adenosine nucleobase in the F8 gene leads to an increase in function of factor VIII protein transcribed from the F8 gene. In some embodiments, deaminating the adenosine nucleobase in the F8 gene leads to an increase in factor VIII stability or half life, for example by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.

In some embodiments, the F8 gene is in a cell, such as a cell in culture or a cell in a subject. In some embodiments, the F8 gene encodes a factor VIII protein comprising a R to C mutation. In some embodiments, the factor VIII protein comprises a R to C mutation (R612C) at residue 612 of the amino acid sequence SEQ ID NO: 341, where the R at position 612 is shown in bold:

(SEQ ID NO: 341) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARF PPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAE VYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKV FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHT FLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYV KVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRS VAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSD KRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSIN GYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDT LTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTG DYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEND IEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYE TFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKL GTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVH YDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWG KNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATN RKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATAL RLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARW IQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQ ENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQ NFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLT QIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPI YLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLE MTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHI YQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRV ATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTI LSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQ REITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEE DQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITA SGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQG ARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAIS DAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in a cell of a subject. In some embodiments, the subject has or is suspected of having hemophilia. In some embodiments, the subject has or is suspected of having hemophilia A. In some embodiments, the subject has a mutation in an F8 gene, where a wild-type G that is mutated to an A, which causes a R to C mutation, for example at amino acid residue 612 (R612C) of SEQ ID NO: 341. In some embodiments, this mutation causes hemophilia (e.g., hemophilia A). In some embodiments, deaminating the adenosine nucleobase in the F8 gene ameliorates one or more symptoms of the hemophilia in the subject.

In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder. In some embodiments, the activity of the fusion protein (e.g., comprising an adenosine deaminase and a Cas9 domain), or the complex, results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a G→A or C→T point mutation associated with a disease or disorder, and wherein the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein, and the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant A results in the codon encoding the wild-type amino acid. In some embodiments, the contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder.

Some embodiments provide methods for using the DNA editing fusion proteins provided herein. In some embodiments, the fusion protein is used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., an A residue. In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or disorder, e.g., hereditary hemochromatosis. In some embodiments, the methods provided herein are used to introduce a point mutation into a promoter of a gene (e.g., a HBG1 or HBG2) that modulates (e.g. increases or decreases) expression of the gene. For example, in some embodiments, methods are provided herein that employ a DNA editing fusion protein to introduce a point mutation into a promoter region of HBG1 or HBG2 to increase expression of HBG1 or HBG2. Such point mutations may, in some embodiments, increase expression of HBG1 or HBG2 in a subject, which may be useful for treating β-globin diseases including sickle-cell anemia.

In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing. The nucleobase editing proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins comprising a nucleic acid programmable DNA binding protein (e.g., Cas9) and an adenosine deaminase domain can be used to correct any single point G to A or C to T mutation. In the first case, deamination of the mutant A to I corrects the mutation, and in the latter case, deamination of the A that is base-paired with the mutant T, followed by a round of replication or followed by base editing repair activity, corrects the mutation.

The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., anemia, or hemochromatosis, an effective amount of an adenosine deaminase fusion protein that corrects a point mutation in a gene (e.g., HFE) or introduces a mutation into a promoter region of a gene (e.g., a promoter region of HBG1 or HBG2). In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a disease associated with anemia. In some embodiments, the disease is an iron storage disease (e.g., hereditary hemochromatosis). In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.

In some embodiments, a fusion protein recognizes canonical PAMs and therefore can correct the pathogenic G to A or C to T mutations with canonical PAMs, e.g., NGG, respectively, in the flanking sequences. For example, Cas9 proteins that recognize canonical PAMs comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 52, or to a fragment thereof comprising the RuvC and HNH domains of SEQ ID NO: 52.

It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and an adenosine deaminase, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. In some embodiments, the guide RNA comprises a structure 5′-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuu uuu-3′ (SEQ ID NO: 389), wherein the guide sequence comprises a sequence that is complementary to the target sequence. In some embodiments, the guide sequence comprises any of the nucleotide sequences provided in FIG. 17 . The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are shown in FIG. 18 , including their locus.

Base Editor Efficiency

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples. in some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively.

In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, a intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described in the “Base Editor Efficiency” section, herein, may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.

Methods for Editing Nucleic Acids

Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to an adenosine deaminase) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the first nucleobase is an adenine. In some embodiments, the second nucleobase is a deaminated adenine, or inosine. In some embodiments, the third nucleobase is a thymine. In some embodiments, the fourth nucleobase is a cytosine. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., A:T to G:C). In some embodiments, the fifth nucleobase is a guanine. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base paires are edited.

In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is adenine, and the second base is not a G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the first base is adenine. In some embodiments, the second base is not a G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window.

In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the first base is adenine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects (e.g., form base excision repair) or binds the non-edited strand. In some embodiments, the nucleobase editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the nucleobase editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein.

Pharmaceutical Compositions

Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the adenosine deaminases, fusion proteins, or the fusion protein-gRNA complexes described herein. The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).

As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.

In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Kits, Vectors, Cells

Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding an adenosine deaminase capable of deaminating an adenosine in a deoxyribonucleic acid (DNA) molecule. In some embodiments, the nucleotide sequence encodes any of the adenosine deaminases provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the adenosine deaminase.

Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to an adenosine deaminase, or a fusion protein comprising a napDNAbp (e.g., Cas9 domain) and an adenosine deaminase as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide nucleic acid backbone, (e.g., a guide RNA backbone), wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide nucleic acid (e.g., guide RNA backbone).

Some aspects of this disclosure provide cells comprising any of the adenosine deaminases, fusion proteins, or complexes provided herein. In some embodiments, the cells comprise a nucleotide that encodes any of the adenosine deaminases or fusion proteins provided herein. In some embodiments, the cells comprise any of the nucleotides or vectors provided herein.

The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

It should be appreciated however, that additional fusion proteins would be apparent to the skilled artisan based on the present disclosure and knowledge in the art.

The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention.

EXAMPLES

Data provided in the below examples describe engineering of base editors that are capable of catalyzing hydrolytic deamination of adenosine (forming inosine, which base pairs like guanine (G)) in the context of DNA. There are no known naturally occurring adenosine deaminases that act on DNA. Instead, known adenosine deaminases act on RNA (e.g., tRNA or mRNA). The first deoxyadenosine deaminases were evolved to accept DNA substrates and deaminate deoxyadenosine (dA) to deoxyinosine. As one example, evolution experiments were performed using the adenosine deaminase acting on tRNA (ADAT) from Escherichia coli (TadA, for tRNA adenosine deaminase A), to engineer adenosine deaminases that act on DNA. Briefly, ecTadA was covalently fused to a dCas9 domain, and libraries of this fusion were assembled containing mutations in the deaminase portion of the construct. In the evolution experiments described below, several mutations in ecTadA were found to improve the ability of ecTadA to deaminate adenosine in DNA. Here the directed evolution, engineering, and characterization of an adenine base editor (ABE) that mediates the programmable conversion of A•T to G•C base pairs in bacterial and human cells is reported. Indeed, approximately half of known pathogenic single nucleotide polymorphisms are C•G to T•A transitions. The ability to convert A•T base pairs to G•C base pairs in a programmable, efficient, and precise manner therefore could substantially advance efforts to study and treat genetic diseases. Extensive evolution and engineering to maximize ABE efficiency and sequence generality resulted in seventh-generation adenine base editors, such as ABE7.10, that convert target A•T to G•C base pairs efficiently (averaging 53% across 17 genomic sites in human cells) with very high product purity (typically >99%) and very low rates of indels comparable to those of untreated cells (typically ≤0.1%). It is shown in the examples that follow that ABET variants introduce point mutations much more efficiently and cleanly than a current Cas9 nuclease-mediated HDR method, induce less off-target genome modification than Cas9 nuclease, and can be used both to correct disease-associated SNPs, and to introduce disease-suppressing SNPs in cultured human cells.

The formation of uracil and thymine from the spontaneous hydrolytic deamination of cytosine and 5-methylcytosine, respectively,^(1,2) occurs an estimated 100-500 times per cell per day in humans¹ and can result in C•G to T•A mutations, accounting for approximately half of all known pathogenic SNPs (FIG. 1A). The ability to convert A•T base pairs to G•C base pairs at target loci in the genomic DNA of unmodified cells therefore could enable the correction of a substantial fraction of human SNPs associated with disease

Base editing is a form of genome editing that enables the direct, irreversible conversion of one base pair to another at a target genomic locus without requiring double-stranded DNA breaks (DSBs), homology-directed repair (HDR) processes, or donor DNA templates.³⁻⁵. Compared with standard genome editing methods to introduce point mutations, base editing can proceed more efficiently⁶, and with far fewer undesired products such as stochastic insertions or deletions (indels) or translocations.^(4,6-8)

The most commonly used base editors are third-generation designs (BE3) that consist of (i) a catalytically impaired CRISPR-Cas9 mutant that cannot make DSBs, (ii) a single-strand-specific cytidine deaminase that converts C to uracil (U) within a small window (˜5 nucleotides) in the single-stranded DNA bubble created by Cas9, (iii) a uracil glycosylase inhibitor (UGI) that impedes uracil excision and downstream processes that decrease base editing efficiency and product purity⁹, and (iv) nickase activity to nick the non-edited DNA strand, directing cellular mismatch repair to replace the G-containing DNA strand^(6,9). Together, these components enable efficient and permanent C•G to T•A base pair conversion in bacteria, yeast^(4,10) plants^(11,12), zebrafish¹³, mammalian cells^(14,15), mice^(16,17), and even human embryos.^(18,19) Base editing capabilities have expanded through the development of base editors with different protospacer-adjacent motif (PAM) compatibilities, narrowed editing windows⁷, enhanced DNA specificity⁸, and small-molecule dependence²⁰. Fourth-generation base editors (BE4 and BE4-Gam) further improve C•G to T•A editing efficiency and product purity.⁹

To date, all reported base editors mediate C•G to T•A conversion. In this study, protein evolution and engineering were used to develop a new class of adenine base editors (ABEs) that convert A•T to G•C base pairs in DNA in bacteria and human cells. Seventh-generation ABEs such as ABE7.10 (FIG. 7 ) convert A•T to G•C at a wide range of target genomic loci in human cells with a typical efficiency of ˜50% and with a very high degree of product purity (>99%), exceeding the typical performance characteristics of BE3. ABEs greatly expand the scope of base editing and, together with previously described base editors, enable the programmable installation of all four transition mutations (C to T, A to G, T to C, and G to A) in the genomes of living cells.

Example 1—Evolution of an Adenine Deaminase that Operates on DNA

The hydrolytic deamination of adenosine yields inosine (FIG. 1B). Within the constraints of a polymerase active site, inosine pairs most stably with C and therefore is read or replicated as G²¹. While replacing the cytidine deaminase domain of an existing base editor with an adenine deaminase could, in theory, provide an ABE (FIG. 1C), no enzymes are known to deaminate adenine in DNA. Although all reported examples of adenine deaminases process either free adenine, free adenosine, adenosine in RNA or in mispaired RNA:DNA heteroduplexes,²² or, curiously, catalyze C to U formation on single-stranded DNA²³, the present efforts were begun by replacing the APOBEC1 component of BE3 with natural adenine deaminases including E. coli TadA²⁴⁻²⁶, human ADAR^(27,28), mouse ADA²⁹, and human ADAT2^(30,31) (Supplementary Sequences 1) to test the possibility that a high effective molarity of single-stranded DNA might overcome their poor activity on DNA. Unfortunately, when plasmid DNA constructs encoding these adenine deaminase-Cas9 D10A nickase fusions were transfected into HEK293T cells together with a corresponding single-guide RNA (sgRNA), no significant A•T to G•C editing was observed above that of untreated cells (FIG. 8A). These results suggest that the inability of the natural adenine deaminase enzymes tested to process DNA precludes their direct use in an ABE.

Given these results, an adenine deaminase variant that accepts DNA as a substrate starting from a naturally occurring RNA adenine deaminase was sought to be evolved. A bacterial selection for base editing was developed by creating antibiotic resistance genes that contain point mutations at critical positions (FIG. 23 and Supplementary Sequences 2). Reversion of these mutations by base editors enables bacterial survival in the presence of antibiotic. To validate the selection, a bacterial codon optimized version of BE2⁶ (APOBEC1 cytidine deaminase fused to dCas9 and UGI) was used, since bacteria lack nick-directed mismatch repair machinery that enables more efficient base editing by BE3³². After optimizing target mutation choice, promoter strength, selection plasmid copy number, incubation times, and antibiotic selection stringency, successful rescue of a defective chloramphenicol acetyl transferase (Cam^(R)) containing an A•T to G•C mutation at a catalytic residue (H193R) by BE2 and a sgRNA programmed to direct base editing to the inactivating mutation was observed, resulting in a chloramphenicol minimum inhibitory concentration (MIC) increase from 1 μg/mL to 32 μg/mL. DNA sequencing confirmed that bacterial cells surviving selection contained the C•G to T•A mutation restoring Cam^(R) function.

Next the selection plasmid was adapted for ABE activity by introducing a C•G to T•A mutation in the Cam^(R) gene, creating an H193Y substitution (FIG. 23 and Supplementary Sequences 2) that confers an MIC of 1 μg/mL chloramphenicol. A•T to G•C conversion at the H193Y mutation should restore chloramphenicol resistance, thereby linking ABE activity to bacterial survival in the presence of chloramphenicol.

The previously described base editors⁶⁻⁹ exploit the use of cytidine deaminase enzymes that operate on single-stranded DNA and reject double-stranded DNA. This single-stranded DNA requirement is critical to focus deaminase activity on a small window of nucleotides within the single-stranded bubble created by Cas9, minimizing undesired deamination events beyond the target nucleotide(s). TadA is a tRNA adenine deaminase²⁴ that converts adenine to inosine (I) in the single-stranded anticodon loop of tRNA^(Arg) . E. coli TadA shares homology with the APOBEC family of enzymes³³ used in the original base editors, and structural studies revealed that some ABOBECs bind single-stranded DNA in a conformation that resembles that of tRNA bound to TadA³³. TadA does not require small-molecule activators (in contrast with ADAR³⁴) and acts on polynucleic acid (unlike ADA²⁹). Based on these considerations, E. coli TadA was chosen as the starting point of the efforts to evolve a DNA adenine deaminase.

Unbiased plasmid libraries of ecTadA-dCas9 fusions containing mutations only in the adenine deaminase portion of the construct to avoid altering the favorable properties of the Cas9 portion of the editor were created. The resulting plasmid libraries were transformed into E. coli harboring the Cam^(R) H193Y selection and ˜5.0×10⁶ transformants were plated on media containing 2 to 16 μg/mL chloramphenicol (FIG. 2A). Surviving colonies were strongly enriched for TadA mutations A106V and D108N (FIG. 2B). Sequence alignment of the evolved E. coli TadA with S. aureus TadA, a homolog for which a structure complexed with tRNA^(Arg) has been reported³⁵, predicts that the side-chain of D108 makes a hydrogen bond with the 2′-OH group of the ribose in the uridine nucleotide immediately upstream of the substrate adenosine (FIG. 2C). Mutations at D108 likely abrogate this hydrogen bond and thereby decrease the energetic opportunity cost of accepting DNA in the substrate-binding site. DNA sequencing confirmed that all bacterial clones surviving the selection showed substantial A•T to G•C reversion at the targeted site in Cam^(R). Collectively, these results indicate that mutations at or near TadA D108 enable TadA to perform adenine deamination on DNA substrates.

The TadA A106V and D108N mutations were incorporated into a mammalian codon-optimized TadA-Cas9 nickase fusion construct that replaces the dCas9 used in bacterial evolution with the Cas9 D10A nickase used in BE3 to manipulate cellular DNA mismatch repair to favor desired base editing outcomes, and adds a C-terminal nuclear localization signal (NLS). Rhe resulting TadA*-XTEN-nCas9-NLS construct, where TadA* represents an evolved TadA variant and XTEN is a 16-amino acid linker used in BE3⁶, was designated as ABE1.2. Transfection of plasmids expressing ABE1.2 and sgRNAs targeting six human genomic sites (FIG. 3A) resulted in very low, but observable A to G editing efficiencies (3.2±0.88%; all editing efficiencies are reported as mean±SD of three biological replicates without enrichment for transfected cells unless otherwise noted) across six diverse target sites in the human genome (FIG. 2A) at or near protospacer position 5, counting the PAM as positions 21-23 (FIG. 3B). These data confirmed that an ABE capable of catalyzing low levels of A•T to G•C conversion emerged from the first round of protein evolution and engineering.

Example 2—Improved Deaminase Variants and ABE Architectures

The editing efficiency of ABE1.2 was sought to be improved through a second round of evolution. An unbiased library of ABE1.2 variants was generated as before, and the resulting TadA*1.2-dCas9 mutants were challenged in bacteria with higher concentrations of chloramphenicol (16 to 128 μg/mL) than was used in round 1 (FIGS. 22 and 23 ). From this second round of evolution, two mutations, D147Y and E155V, predicted to lie in a helix adjacent to the substrate in TadA, were identified (FIG. 2C). In mammalian cells, ABE2.1 (ABE1.2+D147Y+E155V) exhibited 2- to 7-fold higher activity than ABE1.2 at the six genomic sites tested, resulting in an average of 11±2.9% A•T to G•C base editing (FIG. 3B).

Next, ABE2.1 editing efficiencies were sought to be improved through additional protein engineering. Fusing the TadA(2.1)* domain to the C-terminus of Cas9 nickase, instead of the N-terminus, resulted in the complete loss of editing activity (FIGS. 7 and 8B), consistent with previous findings with BE3⁶. Linker lengths were also varied between TadA(2.1)* and Cas9 nickase. An ABE2 variant (ABE2.6) with a linker twice as long (32 amino acids, (SGGS)₂-XTEN-(SGGS)₂,) as the original 16-residue XTEN linker in ABE2.1 offered modestly higher editing efficiencies compared with ABE2.1, now averaging 14±2.4% across the six genomic loci tested (FIGS. 7 and 8B).

Analogous to the mechanism by which uracil N-glycosylase (UNG) catalyzes the removal of uracil from DNA and initiates base excision repair, alkyl adenine DNA glycosylase (AAG) catalyzes the cleavage of the glycosidic bond of inosine in DNA^(36,37). To test if inosine excision impedes ABE performance, ABE2 variants designed to minimize potential sources of inosine base excision repair (BER) were created. Given the absence of known protein inhibitors of AAG, endogenous AAG was attempted to be blocked from accessing the inosine intermediate by separately fusing to the C-terminus of ABE2.1 catalytically inactivated versions of enzymes involved in inosine binding or removal: human AAG (inactivated with a E125Q mutation³⁸), or E. coli Endo V (inactivated with a D35A mutation³⁹). Neither ABE2.1-AAG(E125Q) (ABE2.2) nor ABE2.1-Endo V(D35A) (ABE2.3) exhibited altered A•T to G•C editing efficiencies in HEK293T cells compared with ABE2.1 (FIGS. 7 and 8C). Indeed, using ABE2.1 in Hap1 cells lacking AAG did not result in increases in base editing efficiency, increases in product purity, or decreases in indel frequency compared with Hap1 cells containing wild-type AAG (FIG. 8D). Moreover, ABE2.1 induced virtually no indels (≤0.1%) or A•T to non-G•C products (≤0.1%) at the six loci tested, consistent with inefficient excision of the inosine intermediate (FIGS. 11A to 11B). Taken together, these observations strongly suggest that cellular repair of inosine intermediates created by ABEs is inefficient, obviating the need to subvert processes such as BER. This situation contrasts with that of BE3 and BE4, which are strongly dependent on inhibiting uracil excision to maximize base editing efficiency and product purity, and to suppress indel formation^(6,9).

As a final ABE2 engineering study, the role of the TadA* dimerization state on base editing efficiencies in human cells was explored. In its native form, TadA operates as a homodimer, with one monomer catalyzing A to I deamination, and the other monomer acting as a docking station for the tRNA substrate. During selection in E. coli, it is speculated that endogenous TadA serves as the non-catalytic monomer. In mammalian cells, it is hypothesized that tethering an additional wild-type or evolved TadA monomer might improve editing efficiencies by minimizing reliance on intermolecular ABE dimerization. Indeed, co-expressing with ABE2.1 either wild-type TadA or TadA*2.1 to promote in trans ABE2.1:TadA or ABE2.1:TadA*2.1 dimer formation (ABE2.? and ABE2.8, respectively), as well as direct fusion of either evolved or wild-type TadA to the N-terminus of ABE2.1 (ABE2.9 and ABE2.10, respectively), substantially improved editing efficiencies (FIGS. 3B, 7, and 10A). A fused TadA-ABE2.1 architecture (ABE2.9) was identified to offer the highest editing efficiencies (averaging 20±3.8% across the six genomic loci, and a 7.6±2.6-fold average improvement at each site over ABE1.2) and was used in all subsequent experiments (FIGS. 2B and 3B). A control ABE variant containing two wild-type TadA domains and no evolved TadA* domains did not result in A•T to G•C editing at the six genomic sites tested (FIG. 10A), confirming that dimerization alone was insufficient to mediate ABE activity.

Since these results implicated TadA dimerization as an important component of ABE editing efficiency, it was determined which of the two TadA subunits within the TadA-ABE2 fusion was responsible for A to I catalysis. An inactivating E59A mutation²⁴ was introduced into either the N-terminal or the internal TadA monomer of ABE2.9, generating ABE2.11 or ABE2.12, respectively. The variant with an inactivated N-terminal TadA subunit (ABE2.11) demonstrated comparable editing efficiencies to ABE2, whereas the variant with an inactivated internal TadA subunit lost all editing activity (FIGS. 7 and 10A). These results establish that the internal TadA subunit is responsible for A to I catalysis.

Example 3—ABEs that Efficiently Edit a Subset of Targets

Next, a third round of bacterial evolution was performed starting with TadA*2.1-dCas9 (relying on in trans dimerization with endogenous E. coli TadA or with itself) to further increase A•T to G•C editing efficiencies. The stringency of the selection was increased by introducing two early stop codons (Q4stop and W15stop) in the kanamycin resistance gene (Kan^(R), aminoglycoside phosphotransferase, FIG. 23 and Supplementary Sequences 2). Each of the mutations required an A•T to G•C reversion to correct the premature stop codon. The MIC for host cells harboring the evolution round 3 selection plasmid was ˜8 μg/mL kanamycin and base editing of both Kan^(R) mutations on the same plasmid is required to restore kanamycin resistance (FIG. 23 ). A library of TadA*2.1-dCas9 variants containing mutations in the TadA domain were subjected to this higher stringency selection in the presence of 16 to 128 μg/mL kanamycin, resulting in the strong enrichment of three new TadA mutations: L84F, H123Y, and I157F. These mutations were imported into the ABE2.9 mammalian construct to generate ABE3.1 (FIG. 2B). In HEK293T cells, ABE3.1 resulted in editing efficiencies averaging 29±2.6% across the six tested sites, a 1.6-fold average increase in A•T to G•C conversion at each site over ABE 2.9, and a 11-fold average improvement over ABE1.2 (FIG. 3C). Longer (64- or 100-amino acid) linkers between the two TadA monomers, or between TadA* and Cas9 nickase, were also tested and negative effects on editing efficiencies compared to ABE3.1 were observed (FIGS. 7 and 10B).

Although ABE3.1-mediated base editing efficiencies were high at some sites, such as site 1 (65±4.2% conversion), which placed the edited A at protospacer position 5 in a CAC context, for other sites, such as site 5, which placed the edited A in a GAG context, editing efficiencies were much lower (8.3±0.67% conversion) (FIG. 3C). The results from six genomic loci with different sequence contexts surrounding the target A suggest that ABEs from rounds 1-3 strongly preferred target sequence contexts of YAC, where Y=T or C. This sequence context preference was likely inherited from the substrate specificity of native E. coli TadA, which deaminates the A in the UAC context of the anticodon loop of tRNA^(Arg). The utility of an ABE for base editing applications would be greatly limited, however, by such a target sequence restriction.

To overcome the YAC sequence preference of ABE3.1, a fourth evolution campaign focusing mutagenesis at TadA residues predicted to interact with the nucleotides upstream and downstream of the target A was initiated. Inspection of the S. aureus TadA-tRNA co-crystal structure³⁵ revealed residues that directly contact the anticodon loop of the tRNA substrate, corresponding to E. coli TadA E25, R26, R107, A142, and A143. TadA*2.1-dCas9 libraries (FIG. 22 ) containing randomized residues at these positions were subjected to a new bacterial selection in which A•T to G•C conversion of a non-YAC target (GAT, which causes a T89I mutation in the spectinomycin resistance protein) restores antibiotic resistance (FIG. 23 and Supplementary Sequences 2). Library members in cells harboring the selection plasmid (MIC of ˜32 μg/mL spectinomycin, FIG. 23 ), were challenged with high concentrations of spectinomycin (64 to 512 μg/mL). Surviving bacteria strongly converged on the TadA mutation A142N. Although apparent A•T to G•C base editing efficiency in bacterial cells with TadA*4.3-dCas9 (TadA*3.1+A142N-dCas9) was higher than with TadA*3.1-dCas9 as judged by spectinomycin MIC (FIG. 10C), ABE4.3 generally exhibited decreased base editing efficiencies (averaging 16±5.8%) compared with ABE3.1 in mammalian cells (FIGS. 2B and 3C). It was hypothesized that the A142N mutation may benefit base editing in a context-dependent manner, and revisited its inclusion in later rounds of evolution (see below).

A fifth round of evolution was performed to increase ABE catalytic performance and broaden target sequence compatibility. A library of TadA*3.1-dCas9 variants was generated containing unbiased mutations throughout the TadA* domain as before (FIG. 22 ). To favor ABE constructs with faster kinetics, this library was subjected to the Cam^(R) H193Y selection with higher doses of chloramphenicol (≥128m/mL) after allowing ABE expression for only half the duration (7 h) of the previous rounds of evolution (˜14 h). Surviving clones contained a variety of mutations near the N- and C-terminal domain of TadA. Surprisingly, importing a consensus set of these mutations (H36L, R51L, S146C, and K157N) into ABE3.1, creating ABE5.1, decreased overall editing efficiencies in HEK293T cells by 1.7±0.29-fold (FIGS. 2B and 3C).

Since ABE5.1 included seven mutations since the previous dimerization state experiments on ABE2.1, it was speculated that the accumulation of these new mutations may impair the ability of the non-catalytic N-terminal TadA subunit to play its structural role in mammalian cells. In E. coli, endogenous wild-type TadA monomer is provided in trans, potentially explaining the disconnect between bacterial selection phenotypes and mammalian cell editing efficiencies. Therefore, the effect of using wild-type TadA instead of evolved TadA* variants in the N-terminal, non-catalytic TadA domain of ABE5 variants was examined. These studies revealed that a heterodimeric construct comprised of a wild-type E. coli TadA fused to an internal evolved TadA* (ABE5.3) exhibited greatly improved editing efficiencies compared to the homodimeric ABE5.1 with two identical evolved TadA* domains. ABE5.3 editing efficiencies across the six genomic test sites averaged 39±5.9%, with an average improvement at each site of 2.9±0.78-fold relative to ABE5.1 (FIGS. 2B and 3C). Compared with ABE3.1, ABE5.3 increased editing efficiencies by an average of 1.8±0.39-fold at each tested site. Importantly, ABE5.3 also showed broadened sequence compatibility that now enabled 22-33% editing of non-YAC targets including site 3 (AAG), site 4 (CAA), site 5 (GAG), and site 6 (GAC) (FIG. 3C).

Concurrently, a round 5 library was subjected to the non-YAC spectinomycin selection used in round 4. Although no highly enriched mutations emerged, new mutations from two genotypes emerging from this selection, N72D+G125A; and P48S+S97C (FIG. 7 ), were included in subsequent library generation steps. The simple addition of these mutations to ABE3.1 (generating ABE5.13 and ABE5.14, respectively) did not improve editing efficiencies (FIGS. 7 and 11A).

Since the ABE3 linker studies demonstrated that linkers much longer than 32 amino acids decreased ABE activity (FIGS. 7 and 10B), a more refined approach was taken to optimize ABE5 linkers. Eight heterodimeric wild-type TadA-TadA* ABE5.3 variants (ABE5.5 to ABE5.12) containing 24-, 32-, or 40-residue linkers between the TadA domains or between TadA and Cas9 nickase were tested in HEK293T cells, resulting in no obvious improvements in base editing efficiency (FIGS. 7 and 11B). All subsequent studies thus used the ABE5.3 architecture containing a heterodimeric wtTadA-TadA*-Cas9 nickase with two 32-residue linkers.

Example 4—Highly Active ABEs with Broad Sequence Compatibility

A sixth round of evolution aimed to remove any non-beneficial mutations by DNA shuffling and to reexamine mutations from previous rounds of evolution that may have different effects on ABE performance once liberated from negative epistasis with other mutations. Evolved TadA*-dCas9 variants from rounds 1 through 5 along with wild-type E. coli TadA were shuffled, transformed into E. coli harboring the spectinomycin resistance T89I selection plasmid, and selected on media supplemented with 384 μg/mL spectinomycin. Two mutations were strongly enriched from this selection: P48S/T and A142N (first seen from round 4). These mutations were added either separately or together to ABE5.3, forming ABE6.1 to ABE6.6 (FIG. 7 ). ABE6.3 (ABE5.3+P48S) resulted in 1.3±0.28-fold higher average A•T to G•C editing relative to ABE5.3 at each of the six genomic sites tested, and an average conversion efficiency of 47±5.8% (FIGS. 2B and 4A). P48 is predicted to lie ˜5 A from the substrate adenine nucleobase and 2′-hydroxyl in the TadA crystal structure (FIG. 2C), and it was speculated that mutating this residue to Ser may improve compatibility with a deoxyadenosine substrate. While at most sites ABE6 variants that contained the A142N mutation were less active than ABEs that lack this mutation, editing by ABE6.4 (ABE6.3+A142N) at site 6, which contains a target A at position 7 in the protospacer, was 1.5±0.13-fold more efficient than editing by ABE6.3, and 1.8±0.16-fold more efficient than editing by ABE5.3 (FIG. 4A). These results suggest that A142N variants may offer improved editing of target adenines closer to the PAM than position 5.

Although six rounds of evolution and engineering yielded substantial improvements, ABE6 editors still suffered from reduced editing efficiencies (˜20-40%) at target sequences containing multiple adenines near the targeted A, such as site 3 (AAG) and site 4 (CAA) (FIG. 4A). To address this challenge, a seventh round of evolution was performed in which freshly generated libraries of TadA*6-dCas9 variants were targeted to two separate sites in the kanamycin resistance gene: the Q4stop mutation used in round 3 that requires editing a TAT motif, and a new D208N mutation that requires editing a TAA sequence (FIGS. 22 and 23 , Supplementary Sequences 2). The MIC of host cells harboring the round 7 selection plasmid was 8 μg/mL kanamycin (FIG. 23 ). Unbiased libraries of mutated TadA*6-dCas9 variants were transformed into E. coli, and selected on media containing 64 μg/mL to 384 μg/mL kanamycin. Surviving clones contained three enriched sets of mutations: W23L/R, P48A, and R152H/P.

Introducing these mutations separately or in combinations into mammalian cell ABEs (ABE7.1 to ABE7.10), resulted in substantial increases in A•T to G•C editing efficiencies, especially at targets that contain multiple A residues (FIGS. 2B, 4A, 4B, 7, 12A, and 12B). ABE7.10 edited the six genomic test sites with an average efficiency 58±4.0%, and an average improvement at each site of 1.3±0.20-fold relative to ABE6.3 (FIG. 4A), and 29±7.4-fold compared to ABE1.2. Although mutational dissection revealed that all three of the new mutations contribute to the increase in editing efficiencies (FIGS. 7, 12A, and 12B), the R152P substitution is particularly noteworthy. The aligned ecTadA crystal structure predicts that R152 is in the C-terminal helix and contacts the C in the UAC anticodon loop of the tRNA substrate (FIGS. 2B and 2C). It is speculated that substitution of Arg for Pro disrupts this helix and may abrogate base-specific enzyme:DNA interactions.

Example 5—Characterization of Late-Stage ABEs

The most promising ABEs from rounds 5-7 were characterized in-depth. An expanded set of 17 human genomic targets was chosen that place a target A at position 5 or 7 of the protospacer and collectively include all possible NAN sequence contexts (FIG. 3A). Overall, strong improvement of A•T to G•C editing efficiency was observed in HEK293T cells during the progression from ABES to ABET variants (FIG. 4B). The base editing efficiency of the most active editor overall, ABE7.10, averaged 53±3.7% at the 17 sites tested, exceeded 50% at 11 of these sites, and ranged from 34-68% (FIGS. 4A and 4B). These efficiencies compare favorably to the typical C•G to T•A editing efficiencies of BE3⁶.

Next it was sought to further characterize the base editing activity window of late-stage ABEs. A human genomic site containing an alternating 5′-A-N-A-N-A-N-3′ sequence that could be targeted with either of two sgRNAs such that an A would be located either at every odd position (site 18) or at every even position (site 19) from 2 to 9 in the protospacer was chosen (FIG. 3A). The resulting editing outcomes (FIG. 5A), together with an analysis of editing efficiencies at every protospacer position across all 19 sites tested (FIG. 5B) suggest that the activity windows of late-stage variants are approximately 4-6 nucleotides wide, from protospacer positions ˜4-7 for ABE7.10, and from positions ˜4-9 for ABE6.3, ABE7.8, and ABE7.9, counting the PAM as positions 21-23 (FIGS. 5A to 5C). It is noted that the precise editing window boundaries can vary in a target-dependent manner (FIG. 16 ), as is the case with BE3 and BE4. ABE7.8, ABE7.9, and ABE7.10 were also tested in U2OS cells at sites 1-6 and similar editing results were observed as in HEK293T cells (FIG. 12C), demonstrating that ABE activity is not limited to HEK293T cells.

Analysis of individual high-throughput DNA sequencing reads from ABE editing at 6 to 17 genomic sites in HEK293T cells reveals that base editing outcomes at nearby adenines within the editing window are not statistically independent events. The average normalized linkage disequilibrium (LD) between nearby target adenines steadily increased as ABE evolution proceeded, such that the normalized LD of ABE1.2, ABE3.1, ABE5.3, and ABE7.10 averaged 0.17±0.12, 0.56±0.27, 0.67±0.25, and 0.94±0.08, respectively (FIGS. 14A and 14B). Therefore, early-stage ABEs edit nearby adenines more independently, while late-stage ABEs edit nearby adenines more processively, and are more likely to edit an A if a nearby A in the same DNA strand is also edited. These findings suggest that during the course of evolution, TadA may have evolved kinetic changes that decrease the likelihood of substrate release before additional As within the editing window are converted, resulting in processivity similar to the behavior of BE3⁶.

In contrast to the formation of C to non-T edits and indels that can arise from BE3-mediated base editing of cytidines, ABEs convert A•T to G•C very cleanly in HEK293T and U2OS cells, with an average of <0.1% indels, similar to that of untreated control cells, and no observed A to non-G editing above that of untreated cells among the 17 genomic NAN sites tested (FIGS. 5C and 16 ). It was recently shown that undesired products of BE3 arise from uracil excision and downstream repair processes⁹. The remarkable product purity of all tested ABE variants compared to BE3 suggests that the activity or abundance of enzymes that remove inosine from DNA may be low compared to the those of UNG, resulting in minimal base excision repair following adenine base editing.

ABE7.10 catalyzed A•T to G•C editing efficiencies were compared to those of a state-of-the-art Cas9 nuclease-mediated HDR method, CORRECT⁴¹. At five genomic loci in HEK293T cells average target mutation frequencies ranging from 0.47% to 4.2% with 3.3% to 10.6% indels were observed using the CORRECT HDR method under optimized 48-h conditions in HEK293T cells (FIG. 6A). At these same five genomic loci, ABE7.10 resulted in average target mutation frequencies of 10-35% after 48 h, and 55-68% after 120 h (FIG. 6A), with <0.1% indels (FIG. 6B). The target mutation:indel ratio averaged 0.43 for CORRECT HDR, and >500 for ABE7.10, representing a >1,000-fold improvement in product selectivity favoring ABE7.10. These results demonstrate that ABE7.10 can introduce A•T to G•C point mutations with much higher efficiency and far fewer undesired products than a current Cas9 nuclease-mediated HDR method.

Next the off-target activity of ABE7 variants was examined. Since no method yet exists to comprehensively profile off-target activity of ABEs, it was assumed that off-target ABE editing primarily occurs at the same off-target sites that are edited when Cas9 nuclease is complexed with a particular guide RNA, as has been observed to be the case with BE3^(6,8,9,42). HEK293T cells were treated with three well-characterized guide RNAs (targeting HEK sites 2, 3, and 4)⁴³ and either Cas9 nuclease or ABE7 variants and sequenced the on-target loci and the 12 most active off-target human genomic loci associated with these guide RNAs as identified by the genome-wide GUIDE-Seq method.⁴³ The efficiency of on-target indels by Cas9 and the efficiency of on-target base editing by ABE7.10 both averaged 54% (FIGS. 17-19 ). Detectable modification (≥0.2% indels) by Cas9 nuclease was observed at nine of the 12 (75%) known off-target loci (FIG. 6C and FIGS. 17-19 ). In contrast, when complexed with the same sgRNAs, ABE7.10, ABE7.9, or ABE 7.8 led to ≥0.2% off-target base editing at only four of the 12 (33%) known Cas9 off-target sites. Moreover, the nine confirmed Cas9 off-target loci were modified with an average efficiency of 14% indels, while the four confirmed ABE off-target loci were modified with an average of only 1.3% A•T to G•C mutation (FIGS. 17-19 ). Although seven of the nine confirmed Cas9 off-target loci contained at least one A within the ABE activity window, three of these seven off-target loci were not detectably edited by ABE7.8, 7.9, or 7.10. Together, these data suggest that ABE7 variants may be less prone to off-target genome modification than Cas9 nuclease, even for off-target sites containing editable As. In addition, there was no detected evidence of ABE-induced A•T to G•C editing outside of on-target or off-target protospacers following ABE treatment.

Example 6—ABE-Mediated Correction and Installation of Mutations Relevant to Human Disease

Finally, the potential of ABEs to correct pathogenic mutations and to introduce disease-suppressing mutations in mammalian cells was tested. Mutations in b-globin genes cause a variety of blood diseases. Humans with the rare benign condition HPFH (hereditary persistence of fetal hemoglobin) are resistant to some b-globin diseases including sickle-cell anemia. In certain patients, this phenotype is mediated by mutations in the promoters of the g-globin genes HBG1 and HBG2 that enable sustained expression of fetal hemoglobin, which is normally silenced in humans around birth^(44,45). An sgRNA was designed that programs ABE to simultaneously mutate −198T to C in the promoter driving HBG1 expression, and −198T to C in the promoter driving HBG2 expression, by placing the target A•T base pair at protospacer position 7. These mutations are known to confer British-type HPFH and enable fetal hemoglobin production in adults⁴⁶. ABE7.10 installed the desired T•A to C•G mutations in the HBG1 and HBG2 promoters with 29% and 30% efficiency, respectively, in HEK293T cells (FIGS. 6C and 14 ).

The iron storage disorder hereditary hemochromatosis (HHC) is an autosomal recessive genetic disorder commonly caused by a G to A mutation at nucleotide 845 in the human HFE gene, resulting in a C282Y mutation in the HFE protein^(47,48). This mutation leads to insufficient production of liver iron hormone hepcidin resulting in excessive intestinal iron absorption and potentially life-threatening elevation of serum ferritin. DNA encoding ABE7.10 and a guide RNA that places the target A at protospacer position 5 was transfected into an immortalized lymphoblastoid cell line (LCL) harboring the HFE C282Y genomic mutation. Due to the extreme resistance of LCL cells to transfection, transfected cells were isolated and editing efficiency was measured by HTS of the resulting genomic DNA. The clean conversion of the Tyr282 to Cys282 codon was observed in 28% of total DNA sequencing reads from transfected cells, with no evidence of undesired editing or indels at the on-target locus (FIG. 6C). Although much additional research is needed to develop these ABE editing strategies into potential future clinical therapies for globinopathies, HHC, and other diseases with a genetic component, these examples collectively demonstrate the potential of ABEs to correct disease-driving mutations, and to install mutations known to suppress genetic disease phenotypes, in human cells.

In summary, seven rounds of evolution and engineering transformed a protein that initially exhibited no ability to deaminate adenine at target loci in DNA (wild-type TadA-dCas9 fusions) into forms that edit DNA weakly (ABE1s and ABE2s), variants that edit limited subsets of sites efficiently (ABE3s, ABE4s, and ABE5s), and, ultimately, highly active adenine base editors with broad sequence compatibility (ABE6s and ABE7s). The development of ABEs greatly expands the capabilities of base editing and the fraction of pathogenic SNPs that can be addressed by genome editing without introducing double-stranded DNA breaks (FIG. 1A). In addition, ABEs can also be used to make precise genetic changes of broad utility, including 63 non-synonymous codon changes, the destruction or creation of start codons, the destruction of premature stop codons, the repair of splicing donor or acceptor sites, and the modification of regulatory sequences. ABE7.10 is recommended for general A•T to G•C base editing. When the target A is at protospacer positions 8-10, ABE7.9, ABE7.8, or ABE6.3 may offer higher editing efficiencies than ABE7.10, although conversion efficiencies at these positions are typically lower than at protospacer positions 4-7. Together with BE3 and BE4⁹, these ABEs advance the field of genome editing by enabling the direct installation of all four transition mutations at target loci in living cells with a minimum of undesired byproducts.

Data Availability.

Expression vectors encoding ABE6.3, ABE7.8, ABE7.9, and ABE7.10 are available from Addgene. High-throughput DNA sequencing data will be deposited in the NCBI Sequence Read Archive.

Methods

General Methods.

DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2x Master Mix (New England BioLabs) unless otherwise noted. All mammalian cell and bacterial plasmids generated in this work were assembled using the USER cloning method as previously described⁴⁹ and starting material gene templates were synthetically accessed as either bacterial or mammalian codon-optimized gBlock Gene Fragments (Integrated DNA Technologies). All sgRNA expression plasmids were constructed by a 1-piece blunt-end ligation of a PCR product containing a variable 20-nt sequence corresponding to the desired sgRNA targeted site. Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in FIG. 20 . All mammalian ABE constructs sgRNA plasmids and bacterial constructs were transformed and stored as glycerol stocks at −80° C. in Machl T1^(R) Competent Cells (Thermo Fisher Scientific), which are recA⁻. Molecular Biology grade, Hyclone water (GE Healthcare Life Sciences) was used in all assays and PCR reactions. All vectors used in evolution experiments and mammalian cell assays were purified using ZympPURE Plasmid Midiprep (Zymo Research Corporation), which includes endotoxin removal. Antibiotics used for either plasmid maintenance or selection during evolution were purchased from Gold Biotechnology.

Generation of Bacterial TadA* Libraries (Evolution Rounds 1-3, 5, and 7).

Briefly, libraries of bacterial ABE constructs were generated by two-piece USER assembly of a PCR product containing a mutagenized E. coli TadA gene and a PCR product containing the remaining portion of the editor plasmid (including the XTEN linker, dCas9, sgRNA, selectable marker, origin of replication, and promoter). Specifically, mutations were introduced into the starting template (FIG. 22 ) in 8×25 μL PCR reactions containing 75 ng-1.2 μg of template using Mutazyme II (Agilent Technologies) following the manufacturer's protocol and primers NMG-823 and 824 (FIG. 21 ). After amplification, the resulting PCR products were pooled and purified from polymerase and reaction buffer using a MinElute PCR Purification Kit (Qiagen). The PCR product was treated with Dpn1 (NEB) at 37° C. for 2 h to digest any residual template plasmid. The desired PCR product was subsequently purified by gel electrophoresis using a 1% agarose gel containing 0.5 μg/mL ethidium bromide. The PCR product was extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen) and eluted with 30 μL of H₂O. Following gel purification, the mutagenized ecTadA DNA fragment was amplified with primers NMG-825 and NMG-826 (FIG. 21 ) using Phusion U Green Multiplex PCR Master Mix (8×50 μL PCR reactions, 66° C. annealing, 20-s extension) in order to install the appropriate USER junction sequences onto the 5′ and 3′ end of the fragment. The resulting PCR product was purified by gel electrophoresis. Next, the backbone of the bacterial base editor plasmid template (FIG. 22 ), was amplified with primers NMG-799 and NMG-824 (FIG. 21 ) and Phusion U Green Multiplex PCR Master Mix (100 μL per well in a 98-well PCR plate, 5-6 plates total, Tm 66° C., 4.5-min extension) following the manufacturer's protocol. Each PCR reaction was combined with 300 mL of PB DNA binding buffer (Qiagen) and 25 mL of the solution was loaded onto a HiBind DNA Midi column (Omega Bio-Tek). Bound DNA was washed with 5 column volumes of PE wash buffer (Qiagen) and the DNA fragment was eluted with 800 μL of H₂O per column. Both DNA fragments were quantified using a NanoDrop 1000 Spectrophotometer (Themo Fisher Scientific).

TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of Dpn1 enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 Mm Tris-acetate, 10 mM magnesium acetate, 100m/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). Generally, each round of evolution required ˜1 mL of USER assembly mixture (22 nmol of each DNA assembly fragment) which was distributed into 10-μL aliquots across multiple 8-well PCR strips. The reactions were warmed to 37° C. for 60 min, then heated to 80° C. for 3 min to denature the two enzymes. The assembly mixture was slowly cooled to 12° C. at 0.1° C./s in a thermocycler to promote annealing of the freshly generated ends of the two USER junctions.

With a library of constructs in hand, denatured enzymes and reaction buffer were removed from the assembly mixture by adding 5 vol of PB buffer (Qiagen) to the assembly reaction mixture and binding the material onto a MinElute column (480 μL per column). ABE hybridized library constructs were eluted in 30 μL of H₂O per column and 2 μL of this eluted material was added to 20 μL of NEB 10-beta electrocompotent E. coli and electroporated with a Lonza 4D-Nucleofector System using bacterial program 5 in a 16-well Nucleocuvette strip. A typical round of evolution used ˜300 electroporations to generate 5-10 million colony forming units (cfu). Freshly electroporated E. coli were recovered in 200 mL pre-warmed Davis Rich Media (DRM) at 37° C., and incubated with shaking at 200 rpm in a 500-mL vented baffled flask for 15 min before carbenicillin (for plasmid maintenance) was added to 30m/mL. The culture was incubated at 37° C. with shaking at 200 rpm for 18 h. The plasmid library was isolated with a ZympPURE Plasmid Midiprep kit following manufacturer's procedure (50 mL culture per DNA column), except the plasmid library was eluted in 200 μL pre-warmed water per column. Evolution rounds 1-3, 5 and 7 followed this procedure in order to generate the corresponding library with minor variations (FIG. 22 ).

Generation of Site-Saturated Bacterial TadA* Library (Evolution Round 4).

Mutagenesis at Arg24, Glu25, Arg107, Ala142, and Ala143 of ecTadA was achieved using ecTadA*(2.1)-dCas9 as a template and amplifying with appropriately designed degenerate NNK-containing primers (FIG. 21 ). Briefly, ecTadA*(2.1)-dCas9 template was amplified separately with two sets of primers: NMG-1197+NMG-1200, and NMG-1199+NMG-1200, using Phusion U Green Multiplex PCR Master Mix, forming PCR product 1 and PCR product 2 respectively. Both PCR products were purified individually using PB binding buffer and a MiniElute column and eluted with 20 μL of H₂O per 200 μL of PCR reaction. In a third PCR reaction, 1 μL of PCR product 1 and 1 μL PCR product 2 were combined with exterior, uracil-containing primers NMG-1202 and NMG-1197, and amplified by Phusion U Green Multiplex PCR Master Mix to form the desired extension-overlap PCR product with flanking uracil-containing USER junctions. In a fourth PCR reaction, ecTadA*(2.1)-dCas9 was amplified with NMG-1201 and NMG-1198 to generate the backbone DNA fragment for USER assembly. After Dpn1 digestion and gel purification of both USER assembly fragments, the extension-overlap PCR product (containing the desired NNK mutations in ecTadA) was incorporated into the ecTadA*(2.1)-dCas9 backbone by USER assembly as described above. The freshly generated NNK library was transformed into NEB 10-beta electrocompotent E. coli and the DNA was harvested as described above.

Generation of DNA-Shuffled Bacterial TadA* Library (Evolution Round 6).

DNA shuffling was achieved by a modified version of the nucleotide exchange and excision technology (NExT) DNA shuffling method⁵⁰. Solutions of 10 mM each of dATP, dCTP, dGTP and dTTP/dUTP (3 parts dUTP: 7 parts dTTP) were freshly prepared. Next, the TadA* fragment was amplified from 20 fmol of a pool of TadA*-XTEN-dCas9 bacterial constructs isolated from evolution rounds 1-5 in equimolar concentrations using Taq DNA Polymerase (NEB), primers NMG-822 and NMG-823 (FIG. 21 ), and 400 μM each of dATP, dCTP, dGTP, and dUTP/dTTP (3:7) in 1× ThermoPol Reaction Buffer (Tm 63° C., 1.5-min extension time). The freshly generated uracil-containing DNA library fragment was purified by gel electrophoresis and extracted with QIAquick Gel Extraction Kit (Qiagen), eluting with 20 μL of H₂O per extraction column. The purified DNA product was digested with 2 U of USER enzyme per 40 μL in 1× CutSmart Buffer at 37° C. and monitored by analytical agarose gel electrophoresis until digestion was complete. The reaction was quenched with 10 vol of PN1 binding buffer (Qiagen) when the starting material was no longer observed (typically 3-4 h at 37° C.). Additional USER enzyme was added to the reaction if needed. The digested material was purified with QiaexII kit (Qiagen) using the manufacturer's protocol and the DNA fragments were eluted in 50 μL of pre-warmed H₂O per column.

The purified shuffled TadA* fragment was reassembled into full-length TadA*-XTENdCas9 product by an internal primer extension procedure. The eluted digested DNA fragments (25 μL) were combined with 4 U of Vent Polymerase (NEB), 800 μM each of dATP, dCTP, dGTP, and dTTP, 1 U of Taq DNA polymerase in 1× ThermoPol Buffer supplemented with 0.5 mM MgSO4. The thermocycler program for the reassembly procedure was the following: 94° C. for 3 min, 40 cycles of denaturation at 92° C. for 30 s, annealing over 60 s at increasing temperatures starting at 30° C. and adding 1° C. per cycle (cooling ramp=1° C./s), and extension at 72° C. for 60 s with an additional 4 s per cycle, ending with one final cycle of 72° C. for 10 min. The reassembled product was amplified by PCR with the following conditions: 15 μL of unpurified internal assembly was combined with 1 μM each of USER primers NMG-825 and NMG-826, 100 μL of Phusion U Green Multiplex PCR Master Mix and H₂O to a final volume of 200 μL, 63° C. annealing, extension time of 30 s. The PCR product was purified by gel electrophoresis and assembled using the USER method into the corresponding ecTadA*-XTEN-dCas9 backbone with corresponding flanking USER junctions generated from amplification of the backbone with USER primers NMG-799 and NMG-824 as before. The library of evolution 6 constructs was isolated using a ZymoPURE Plasmid Midiprep kit following the manufacturer's procedure following transformation of the hybridized library into NEB 10-beta electrocompotent E. coli.

Bacterial Evolution of TadA Variants.

The previously described strain S1030⁵¹ was used in all evolution experiments and an electrocompotent version of the bacteria was prepared as previously described⁴⁹ harboring the appropriate selection plasmid specific to each round of evolution (FIG. 22 ). Briefly, 2 μL of freshly generated TadA* library (300-600 ng/μL) prepared as described above was added to 22 μL of freshly prepared electrocompotent S1030 cells containing the target selection plasmid and electroporated with a Lonza 4D-Nucleofector System using bacterial program 5 in a 16-well Nucleocuvette strip. A typical selection used 5-10×10⁶ cfu. After electroporation, freshly transformed S1030 cells were recovered in a total of 250 mL of pre-warmed DRM media at 37° C. shaking at 200 rpm for 15 min. Following this brief recovery incubation, carbenicillin was added to a final concentration of 30m/mL to maintain the library plasmid, along with the appropriate antibiotic to maintain the selection plasmid; see FIG. 22 for the list of selection conditions including the antibiotics used for each round. Immediately following the addition of the plasmid maintenance antibiotics, 100 mM of L-Arabinose was added to the culture to induce translation of TadA*-dCas9 fusion library members, which were expressed from the P_(BAD) promoter. The culture was grown to saturation at 37° C. with shaking at 200 rpm for 18 h, except the incubation time for evolution round 5 was only 7 h).

Library members were challenged by plating 10 mL of the saturated culture onto each of four 500-cm2 square culture dishes containing 1.8% agar-2×YT, 30m/mL of plasmid maintenance antibiotics, and a concentration of the selection antibiotic pre-determined to be above the MIC of the 51030 strain harboring the antibiotic alone (FIG. 23 ). Plates were incubated at 37° C. for 2 days and ˜500 surviving colonies were isolated. The TadA* genes from these colonies were amplified by PCR with primers NMG-822 and NMG-823 (FIG. 21 ) and submitted for DNA sequencing. Concurrently, the colonies were inoculated separately into 1-mL DRM cultures in a 96-deep well plate and grown overnight at 37° C., 200 r.p.m. Aliquots (100 μL) of each overnight culture were pooled, the plasmid DNA was isolated, and the TadA* genes were amplified with USER primers NMG-825 and NMG-826 (FIG. 21 ). The TadA* genes were subcloned back into the plasmid backbone (containing the XTEN linker-dCas9, and appropriate guide RNAs) with the USER assembly protocol described above. This enriched library was transformed into the appropriate S1030 (+selection plasmid) electrocompotent cells, incubated with maintenance antibiotic and L-Ara and re-challenged with the selection condition. After 2-day incubation, 300-400 surviving clones were isolated as described above and their TadA* genes were sequenced. Mutations arising from each selection round were imported into mammalian ABE constructs and tested in mammalian cells as described below.

General Mammalian Cell Culture Conditions.

HEK293T (ATCC CRL-3216) and U2OS (ATTC HTB-96) were purchased from ATCC and cultured and passaged in Dulbecco's Modified Eagle's Medium (DMEM) plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS). Hap1 (Horizon Discovery, C631) and Hap1 AAGcells (Horizon Discovery, HZGHC001537c002) were maintained in Iscove's Modified Dulbecco's Medium (IMDM) plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) FBS. Lymphoblastoid cell lines (LCL) containing a C282Y mutation in the HFE gene (Coriell Biorepository, GM14620) were maintained in Roswell Park Memorial Institute Medium 1640 (RPMI-1640) plus GlutaMax (ThermoFisher Scientific) supplemented with 20% FBS. All cell types were incubated, maintained, and cultured at 37° C. with 5% CO₂.

HEK293T Tissue Culture Transfection Protocol and Genomic DNA Preparation.

HEK293T cells grown in the absence of antibiotic were seeded on 48-well poly-D-lysine coated plates (Corning). 12-14 h post-seeding, cells were transfected at approximately 70% confluency with 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocols and 750 ng of ABE plasmid, 250 ng of sgRNA expression, and 10 ng of a GFP expression plasmid (Lonza). Unless otherwise stated, cells were cultured for 5 days, with a media change on day 3. Media was removed, cells were washed with 1×PBS solution (Thermo Fisher Scientific), and genomic DNA was extracted by addition of 100 μL freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.0, 0.05% SDS, 25 μg/mL Proteinase K (ThermoFisher Scientific) directly into each well of the tissue culture plate. The genomic DNA mixture was transferred to a 96-well PCR plate and incubated at 37° C. for 1 h, followed by an 80° C. enzyme denaturation step for 30 min. Primers used for mammalian cell genomic DNA amplification are listed in FIG. 24 .

Nucleofection of HAP1 and HAP1 AAG⁻ Cells and Genomic DNA Extraction.

HAP1 and HAP1 AAG⁻ cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit S according to the manufacturer's protocol. Briefly, 4×10⁵ cells were nucleofected with 300 ng of ABE plasmid and 100 ng of sgRNA expression plasmid using the 4D-Nucleofector program DZ-113 and cultured in 250 μL of media in a 48-well poly-D-lysine coated culture plate for 3 days. DNA was extracted as described above.

Nucleofection of U2OS Cells and Genomic DNA Extraction.

U2OS cells were nucleofected using the SG Cell Line 4D-Nucleofector X Kit (Lonza) according to the manufacture's protocol. Briefly, 1.25×10⁵ cells were nucleofected in 20 μL of SG buffer along with 500 ng of ABE plasmid and 100 ng of sgRNA expression plasmid using the 4D-Nucleofector program EH-100 in a 16-well Nucleocuvette strip (20 μL of cells per well). Freshly nucleofected cells were transferred into 250 μL of media in a 48-well poly-D-lysine coated culture plate. Cells were incubated for 5 days and media was changed every day. DNA was extracted as described above.

Electroporation of LCL HFE C828Y Cells.

LCL cells were electroporated using a Gene Pulser Xcell Electroporater (BioRad) and 0.4 cm gap Gene Pulser electroporation cuvettes (BioRad). Briefly, 1×10⁷ LCL cells were resuspended in 250 μL RPMI-160 plus GlutaMax. To this media was added 65 μg of plasmid expressing ABE7.10, GFP, and the corresponding sgRNA targeting the C282Y mutation in the HFE gene. The mixture was added to a prechilled 0.4 cm gap electroporation cuvette and the cell/DNA mixture was incubated in the cuvette on ice for 10 min. Cells were pulsed at 250 V and 950 μF for 3 ms. Cells were transferred back on ice for 10 min, then transferred to 15 mL of pre-warmed RPMI-160 supplemented with 20% FBS in a T-75 flask. The next day, an additional 5 mL of media was added to the flask and cells were left to incubate for a total of 5 days. After incubation, cells were isolated by centrifugation, resuspended in 400 μL of media, filtered through a 40 μm strainer (Thermo Fisher Scientific), and sorted for GFP fluorescence using an FACSAria III Flow Cytometer (Becton Dickenson Biosciences). GFP-positive cells were collected in a 1.5-mL tube containing 500 μL of media. After centrifugation, the media was removed and cells were washed twice with 600 μL of 1×PBS (Thermo Fisher Scientific). Genomic DNA was extracted as described above.

Comparison Between ABE 7.10 and Homology Directed Repair Using the ‘CORRECT’ Method⁵².

HEK293T cells grown in the absence of antibiotic were seeded on 48-well poly-Dlysine coated plates (Corning). After 12-14 h, cells were transfected at ˜70% confluency with 750 ng of Cas9 or base editor plasmid, 250 ng of sgRNA expression plasmid, 1.5 μL of Lipofectamine 3000 (Thermo Fisher Scientific), and for HDR assays 0.7 μg of single-stranded donor DNA template (100 nt, PAGE-purified from IDT) according to the manufacturer's instructions. 100-mer single-stranded oligonucleotide donor templates are listed in FIG. 25 .

Genomic DNA was harvested 48 h post-transfection (as described by Tessier-Lavigne et. al. during the development of the CORRECT method52) using the Agencourt DNAdvance Genomic DNA isolation Kit (Beckman Coulter) according to the manufacturer's instructions. A size-selective DNA isolation step ensured that there was no risk of contamination by the single-stranded donor DNA template in subsequent PCR amplification and sequencing steps. Amplification primers were re-designed to ensure there was minimal risk of amplifying donor oligo template.

High-Throughput DNA Sequencing (HTS) of Genomic DNA Samples.

Genomic sites of interest were amplified by PCR with primers containing homology to the region of interest and the appropriate Illumina forward and reverse adapters (FIG. 24 ). Primer pairs used in this first round of PCR (PCR 1) for all genomic sites discussed in this work can be found in FIG. 24 . Specifically, 25 μL of a given PCR 1 reaction was assembled containing 0.5 μM of each forward and reverse primer, 1 μL of genomic DNA extract and 12.5 μL of Phusion U Green Multiplex PCR Master Mix. PCR reactions were carried out as follows: 95° C. for 2 min, then 30 cycles of [95° C. for 15 s, 62° C. for 20 s, and 72° C. for 20 s], followed by a final 72° C. extension for 2 min. PCR products were verified by comparison with DNA standards (Quick-Load 100 bp DNA ladder) on a 2% agarose gel supplemented with ethidium bromide. Unique Illumina barcoding primer pairs were added to each sample in a secondary PCR reaction (PCR 2). Specifically, 25 μL of a given PCR 2 reaction was assembled containing 0.5 μM of each unique forward and reverse illumine barcoding primer pair, 2 μL of unpurified PCR 1 reaction mixture, and 12.5 μL of Q5 Hot Start High-Fidelity 2× Master Mix. The barcoding PCR 2 reactions were carried out as follows: 95° C. for 2 min, then 15 cycles of [95° C. for 15 s, 61° C. for 20 s, and 72° C. for 20 s], followed by a final 72° C. extension for 2 min. PCR products were purified by electrophoresis with a 2% agarose gel using a QIAquick Gel Extraction Kit, eluting with 30 μL of H2O. DNA concentration was quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and sequenced on an Illumina MiSeq instrument according to the manufacturer's protocols.

General HTS Data Analysis.

Sequencing reads were demultiplexed in MiSeq Reporter (Illumina). Alignment of amplicon sequences to a reference sequence was performed as previously described using a Matlab script with improved output format (Supplementary Note 1). In brief, the Smith-Waterman algorithm was used to align sequences without indels to a reference sequence; bases with a quality score less than 30 were converted to ‘N’ to prevent base miscalling as a result of sequencing error. Indels were quantified separately using a modified version of a previously described Matlab script in which sequencing reads with more than half the base calls below a quality score of Q30 were filtered out (Supplementary Note 2). Indels were counted as reads which contained insertions or deletions of greater than or equal to 1 bp within a 30-bp window surrounding the predicted Cas9 cleavage site.

Due to homology in the HBG1 and HBG2 loci, primers were designed that would amplify both loci within a single PCR reaction. In order to computationally separate sequences of these two genomic sites, sequencing experiments involving this amplicon were processed using a separate Python script (Supplementary Note 3). Briefly, reads were disregarded if more than half of the base calls were below Q30, and base calls with a quality score below Q30 were converted to ‘N’. HBG1 or HBG2 reads were identified as having an exact match to a 37-bp sequence containing two SNPs that differ between the sites. A base calling and indel window were defined by exact matches to 10-bp flanking sequences on both sides of a 43-bp window centered on the protospacer sequence. Indels were counted as reads in which this base calling window was ≥1 bp different in length. This Python script yields output with identical quality (estimated base calling error rate of <1 in 1,000), but in far less time due to the absence of an alignment step.

To calculate the total number of edited reads as a proportion of the total number of successfully sequenced reads, the fraction of edited reads as measured by the alignment algorithm were multiplied by [1−fraction of reads containing an indel].

Linkage Disequlilbrium Analysis.

A custom Python script (Supplementary Note 4) was used to assess editing probabilities at the primary target A (P₁) at the secondary target A (P2), and at both the primary and secondary target As (P1,2). Linkage disequilibrium (LD) was then evaluated as P_(1,2)−(P₁×P₂). LD values were normalized with a normalization factor of Min(P₁(1−P₂), (1−P₁)P₂). This normalization which controls for allele frequency and yields a normalized LD value from 0 to 1.

Example 7—ABE-Mediated Installation of Mutations in the HBG1/2 Promoter to Treat Human Disease, Such as Sickle Cell and Beta-Thalassemia

The sgRNAs indicated in FIG. 15 target sites in the HBG1/2 promoters, primarily focused on sites at or near known mutations that confer fetal hemoglobin upregulation in adults. ABE 7.10 and individual sgRNA plasmids (750 ng of editor, 250 ng of guide) were lipofected into HEK293T cells. After three days of expression in HEK293T cells, media was aspirated and fresh media was added. Following two more days of growth, DNA was extracted. PCR was conducted to amplify the HBG1/2 promoter regions. PCR products were sequenced using an Illumina MiSeq, and edits were quantified for each sample. Plotted in FIG. 15 is the highest editing observed within the window for cells treated with the indicated guide. These results establish that mutations can be introduced that could upregulate fetal hemoglobin in adults. The introduction of such mutations could be therapeutic for sickle cell disease and beta thalassemia.

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Supplementary Sequences 1.

DNA sequences of adenine deaminases used in this study.

Bacterial codon-optimized ecTadA (wild-type): (SEQ ID NO: 31) ATGTCTGAAGTCGAATTTAGCCACGAATACTGGATGCGTCACGCGCTGA CGCTGGCGAAACGTGCCTGGGATGAGCGGGAAGTGCCGGTCGGCGCGGT ATTAGTGCATAACAATCGGGTAATCGGCGAAGGCTGGAACCGCCCGATT GGTCGCCATGATCCCACCGCACATGCAGAAATCATGGCCCTGCGGCAGG GTGGTCTGGTGATGCAAAATTATCGTCTGATCGACGCCACGTTGTATGT CACGCTTGAACCATGTGTAATGTGTGCCGGAGCGATGATCCACAGTCGC ATTGGTCGCGTGGTCTTTGGTGCGCGTGACGCGAAAACTGGCGCTGCGG GATCTTTAATGGATGTGCTGCATCATCCGGGTATGAATCACCGAGTGGA AATTACGGAAGGAATACTGGCGGATGAGTGCGCGGCGTTGCTCAGTGAC TTCTTTCGCATGCGCCGCCAGGAAATTAAAGCGCAGAAAAAAGCGCAAT CCTCGACGGAT Mammalian codon-optimized ecTadA (wild-type): (SEQ ID NO: 32) ATGTCCGAAGTCGAGTTTTCCCATGAGTACTGGATGAGACACGCATTGA CTCTCGCAAAGAGGGCTTGGGATGAACGCGAGGTGCCCGTGGGGGCAGT ACTCGTGCATAACAATCGCGTAATCGGCGAAGGTTGGAATAGGCCGATC GGACGCCACGACCCCACTGCACATGCGGAAATCATGGCCCTTCGACAGG GAGGGCTTGTGATGCAGAATTATCGACTTATCGATGCGACGCTGTACGT CACGCTTGAACCTTGCGTAATGTGCGCGGGAGCTATGATTCACTCCCGC ATTGGACGAGTTGTATTCGGTGCCCGCGACGCCAAGACGGGTGCCGCAG GTTCACTGATGGACGTGCTGCATCACCCAGGCATGAACCACCGGGTAGA AATCACAGAAGGCATATTGGCGGACGAATGTGCGGCGCTGTTGTCCGAC TTTTTTCGCATGCGGAGGCAGGAGATCAAGGCCCAGAAAAAAGCACAAT CCTCTACTGAC Mammalian codon-optimized mADA: (SEQ ID NO: 33) ATGGCCCAGACACCCGCATTCAACAAACCCAAAGTAGAGTTACACGTCC ACCTGGATGGAGCCATCAAGCCAGAAACCATCTTATACTTTGGCAAGAA GAGAGGCATCGCCCTCCCGGCAGATACAGTGGAGGAGCTGCGCAACATT ATCGGCATGGACAAGCCCCTCTCGCTCCCAGGCTTCCTGGCCAAGTTTG ACTACTACATGCCTGTGATTGCGGGCTGCAGAGAGGCCATCAAGAGGAT CGCCTACGAGTTTGTGGAGATGAAGGCAAAGGAGGGCGTGGTCTATGTG GAAGTGCGCTATAGCCCACACCTGCTGGCCAATTCCAAGGTGGACCCAA TGCCCTGGAACCAGACTGAAGGGGACGTCACCCCTGATGACGTTGTGGA TCTTGTGAACCAGGGCCTGCAGGAGGGAGAGCAAGCATTTGGCATCAAG GTCCGGTCCATTCTGTGCTGCATGCGCCACCAGCCCAGCTGGTCCCTTG AGGTGTTGGAGCTGTGTAAGAAGTACAATCAGAAGACCGTGGTGGCTAT GGACTTGGCTGGGGATGAGACCATTGAAGGAAGTAGCCTCTTCCCAGGC CACGTGGAAGCCTATGAGGGCGC AGTAAAGAATGGCATTCATCGGACCGTCCACGCTGGCGAGGTGGGCTCT CCTGAGGTTGTGCGTGAGGCTGTGGACATCCTCAAGACAGAGAGGGTGG GACATGGTTATCACACCATCGAGGATGAAGCTCTCTACAACAGACTACT GAAAGAAAACATGCACTTTGAGGTCTGCCCCTGGTCCAGCTACCTCACA GGCGCCTGGGATCCCAAAACGACGCATGCGGTTGTTCGCTTCAAGAATG ATAAGGCCAACTACTCACTCAACACAGACGACCCCCTCATCTTCAAGTC CACCCTAGACACTGACTACCAGATGACCAAGAAAGACATGGGCTTCACT GAGGAGGAGTTCAAGCGACTGAACATCAACGCAGCGAAGTCAAGCTTCC TCCCAGAGGAAGAGAAGAAGGAACTTCTGGAACGGCTCTACAGAGAATA CCAA Mammalian codon optimized hADAR2 (catalytic domain): (SEQ ID NO: 41) ATGCATCTCGATCAAACCCCGAGCCGCCAACCAATCCCGAGTGAAGGCC TGCAACTGCATCTGCCACAAGTTCTGGCGGATGCCGTTAGCCGCCTGGT CTTGGGTAAGTTCGGTGATCTGACAGACAACTTTTCTAGTCCACATGCT CGCCGTAAGGTGCTGGCTGGCGTTGTGATGACCACAGGTACAGACGTCA AAGATGCTAAAGTGATTTCTGTGTCTACTGGCACGAAGTGCATTAACGG CGAATATATGTCTGACCGTGGCTTAGCGCTTAACGATTGTCATGCCGAA ATCATCTCCCGTCGTTCATTGCTTCGCTTCCTGTACACGCAGTTGGAAC TGTATCTGAATAACAAAGACGATCAGAAGCGTTCTATTTTCCAGAAGTC TGAGCGCGGCGGGTTCCGTCTTAAAGAGAATGTGCAGTTTCACCTTTAT ATTTCAACCTCTCCTTGTGGTGATGCCCGTATTTTTTCACCACACGAAC CTATTTTAGAGGAACCGGCCGATCGTCATCCGAACCGCAAAGCCCGTGG GCAGCTGCGTACGAAAATCGAATCAGGTGAAGGCACCATTCCCGTCCGC TCCAATGCGAGCATTCAAACGTGGGACGGTGTGTTACAGGGCGAACGCC TGTTAACCATGAGCTGCTCAGACAAAATTGCACGTTGGAACGTGGTAGG CATCCAGGGCTCGTTATTGAGCATTTTCGTGGAGCCGATTTATTTTAGT TCCATCATTTTGGGCTCACTCTACCACGGCGATCACCTTAGCCGCGCGA TGTACCAGCGCATTAGTAACATCGAAGATTTACCGCCCCTGTATACCCT GAACAAACCACTGTTAAGCGGTATTTCTAACGCGGAGGCGCGTCAGCCT GGTAAAGCCCCGAACTTCAGTGTGAACTGGACTGTGGGTGATTCTGCAA TTGAGGTAATTAACGCGACGACGGGTAAAGATGAACTGGGCCGTGCCTC TCGTCTGTGTAAACACGCGCTGTACTGTCGTTGGATGCGCGTGCACGGT AAAGTTCCCAGTCATCTGTTACGTAGCAAGATCACCAAGCCAAATGTCT ACCACGAATCGAAGCTGGCCGCGAAAGAATACCAAGCGGCTAAGGCGCG TCTGTTCACCGCCTTTATTAAGGCTGGCTTAGGGGCCTGGGTGGAAAAA CCAACCGAGCAAGATCAATTCAGTCTGACCCCG Mammalian codon optimized hADAT2: (SEQ ID NO: 42) ATGGAGGCGAAGGCGGCACCCAAGCCAGCTGCAAGCGGCGCGTGCTCGG TGTCGGCAGAGGAGACCGAAAAGTGGATGGAGGAGGCGATGCACATGGC CAAAGAAGCCCTCGAAAATACTGAAGTTCCTGTTGGCTGTCTTATGGTC TACAACAATGAAGTTGTAGGGAAGGGGAGAAATGAAGTTAACCAAACCA AAAATGCTACTCGACATGCAGAAATGGTGGCCATCGATCAGGTCCTCGA TTGGTGTCGTCAAAGTGGCAAGAGTCCCTCTGAAGTATTTGAACACACT GTGTTGTATGTCACTGTGGAGCCGTGCATTATGTGTGCAGCTGCTCTCC GCCTGATGAAAATCCCGCTGGTTGTATATGGCTGTCAGAATGAACGATT TGGTGGTTGTGGCTCTGTTCTAAATATTGCCTCTGCTGACCTACCAAAC ACTGGGAGACCATTTCAGTGTATCCCTGGATATCGGGCTGAGGAAGCAG TGGAAATGTTAAAGACCTTCTACAAACAAGAAAATCCAAATGCACCAAA ATCGAAAGTTCGGAAAAAGGAATGTCAGAAATCT Supplementary Sequences 2.

DNA sequences of antibiotic resistance genes used in this study. Inactivating mutations are shown in bold.

Chloramphenicol resistance gene (Cam^(R)) H193Y: (SEQ ID NO: 43) ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCA TCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATA ACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAA AATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGAT GAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGA TATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAA ACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCT ACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATT TCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGG GTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTT CGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGC TGATGCCGCTGGCCATCCAGGTGCACTACGCCGTATGCGACGGCTTCCAT GTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGG CGGGGCGTAA Kanamycin resistance gene (Kan^(R)) Q4STOP and W15STOP: (SEQ ID NO: 44) ATGATCGAATAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTAGGTGGA GCGCCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAG ACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCT ATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTG TCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAG GATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGC TGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCG ACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCC AGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATC TCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAAT GGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCG CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCG GCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGAT TCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTAA Spectinomycin resistance gene (Spect^(R)) T89I: (SEQ ID NO: 45) ATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGT TGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGT ACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTG CTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGAT CAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCC GCGCTGTAGAAGTCA T CATTGTTGTGCACGACGACATCATTCCGTGGCGT TATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACAT TCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCT TGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCG GAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAA TGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGC GAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGC AAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCC GGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGAC AAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAA Kanamycin resistance gene (Kan^(R)) Q4STOP and D208N: (SEQ ID NO: 46) ATGATCGAA T AAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGA GAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAG ACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCT ATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTG TCACTGAAGCGGGAAGGGACTGGCTGCTATAGCCGGCCACAGTTAATGAA TGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCC GAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGA TCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAG CACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAA GAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCG CATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGC CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATT A ACTGTGGC CGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGA TATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTT GACGAGTTCTTCTAA Supplementary Note 1.

Matlab script for base calling.

function basecall(WTnuc, directory) %cycle through fastq files for different samples cd directory files=dir(‘*.fastq’); for d=1:2 filename=files(d).name; %read fastq file [header,seqs,qscore] = fastqread(filename); seqsLength = length(seqs); % number of sequences seqsFile = strrep(filename,‘.fastq’,”); % trims off .fastq %create a directory with the same name as fastq file if exist(seqsFile,‘dir’); error(‘Directory already exists. Please rename or move it before moving on.’); end mkdir(seqsFile); % make directory wtLength = length(WTnuc); % length of wildtype sequence %% aligning back to the wildtype nucleotide sequence % % AlN is a matrix of the nucleotide alignment window=1:wtLength; sBLength = length(seqs); % number of sequences % counts number of skips nSkips = 0; ALN=repmat(‘’,[sBLength wtLength]); % iterate through each sequencing read for i = 1:sBLength %If you only have forward read fastq files leave as is %If you have R1 foward and R2 is reverse fastq files uncomment the %next four lines of code and the subsequent end statement % if mod(d,2)==0; % reverse = seqrcomplement(seqs{i}); % [score,alignment,start] = swalign(reverse,WTnuc,‘Alphabet’,‘NT’); % else [score,alignment,start] = swalign(seqs{i},WTnuc,’Alphabet’,’NT’); % end % length of the sequencing read len = length(alignment(3,:)); % if there is a gap in the alignment , skip = 1 and we will % throw away the entire read skip = 0; for j = 1:len if (alignment(3,j) == ‘−’ || alignment(1,j) == ‘−’) skip = 1; break; letters) end %in addition if the qscore for any given base in the read is %below 31 the nucleotide is turned into an N (fastq qscores that are not if isletter(qscore{i}(start(1)+j−1)) else alignment(1,j) = ‘N’; end end if skip == 0 && len>10 ALN(i, start(2):(start(2)+length(alignment)−1))=alignment(1,:); end end % with the alignment matrices we can simply tally up the occurrences of % each nucleotide at each column in the alignment these % tallies ignore bases annotated as N % due to low qscores TallyNTD=zeros(5,wtLength); FreqNTD=zeros(4,wtLength); SUM=zeros(1,wtLength); for i=1:wtLength TallyNTD(:,i)=[sum(ALN(:,i)==‘A’),sum(ALN(:,i)==‘C’),sum(ALN(:,i)==‘G’),sum(ALN(:,i)= =‘T’ ),sum(ALN(:,i)==‘N’)]; end for i=1:wtLength FreqNTD(:,i)=100*TallyNTD(1:4,i)/sum(TallyNTD(1:4,i)); end for i=1:wtLength SUM(:,i)=sum(TallyNTD(1:4,i)); end % we then save these tally matrices in the respective folder for % further processing save(strcat(seqsFile, ‘/TallyNTD’), ‘TallyNTD’); dlmwrite(strcat(seqsFile, ‘/TallyNTD.csv’), TallyNTD, ‘precision’, ‘%.3f’, ‘newline’, ‘pc’); save(strcat(seqsFile, ‘/FreqNTD’), ‘FreqNTD’); dlmwrite(strcat(seqsFile, ‘/FreqNTD.csv’), FreqNTD, ‘precision’, ‘%.3f’, ‘newline’, ‘pc’); fid = fopen(‘FrequencySummary.csv’, ‘a’); fprintf(fid, ‘\n \n’); fprintf(fid, filename); fprintf(fid, ‘\n \n’); dlmwrite(‘FrequencySummary.csv’, FreqNTD, ‘precision’, ‘%.3f’, ‘newline’, ‘pc’,‘- append’); dlmwrite(‘FrequencySummary.csv’, SUM, ‘precision’, ‘%.3f’, ‘newline’, ‘pc’,‘- append’); end % set up queue of basecalling runs % change directory to folder of fastq files for a given target site cd(‘/Users/michaelpacker/Documents/MATLAB/BaseCallingWithSummary’) cd PUTFOLDERNAMEHERE % call upon the basecall program basecall(PUTWTSEQUENCEHERE) % and repeat cd(‘/Users/michaelpacker/Documents/MATLAB/BaseCallingWithSummary’) cd PUTFOLDERNAMEHERE basecall(PUTWTSEQUENCEHERE) % and repeat... Supplementary Note 2.

Matlab script for indel analysis.

%WTnuc=′CGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC CGCTAGAGATCCGCGGCCGCTAATACGACTCAC CCTAGGGAGAGCCGCCACCGTGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAG′; %cycle through fastq files for different samples files=dir(′*.fastq′); indelstart=55; width=30; flank=10; (SEQ ID NO: 85) for d=1:2 filename=files(d).name; %read fastq file [header,seqs,qscore]=fastqread(filename); seqsLength=length(seqs); % number of sequences seqsFile=strcat(strrep(filename,′.fastq′,″),′_INDELS′); % trims off.fastq %create a directory with the same name as fastq file+_INDELS if exist(seqsFile,′dir′); error(′Directory already exists. Please rename or move it before moving on.′); end mkdir(seqsFile); % make directory wtLength=length(WTnuc); % length of wildtype sequence sBLength=length(seqs); % number of sequences % initialize counters and cell arrays nSkips=0; notINDEL=0; ins={}; dels={}; NumIns=0; NumDels=0; % iterate through each sequencing read for i=1:sBLength %search for 10 BP sequences that should flank both sides of the ″INDEL WINDOW″ window start=strfind(seqs{i},WTnuc(indelstart-flank:indelstart)); windowend=strfind(seqs{i},WTnuc(indelstart+width:indelstart+width+flank)); %if these flanks are found and more than half of base calls %are above Q31 THEN proceed OTHERWISE save as a skip if length(windowstart)==1 && length(windowend)==1 && (sum(isletter(qscore{i}))/length(qscore{ii}))>=0.5 %if the sequence length matches the INDEL window length save as %not INDEL if windowend-windowstart==width+flank notINDEL=notINDEL+1; %if the sequence is ONE or more baseslonger than the INDEL %window length save as an Insertion elseif windowend-windowstart>=width+flank+1 NumIns=NumIns+1; ins{NumIns}=seqs{i]; %if the sequence is ONE or more bases shorter than the INDEL %window length save as a Deletion elseif windowend-windowstart<=width+flank-1 NumDels=NumDels+1; dels{NumDels}=seqs{i}; end %keep track of skipped sequences that do not posess matching flank %sequences and do not pass quality cutoff else nSkips=nSkips+1; end end INDELrate=(NumIns+NumDels)/(NumIns+NumDels+notINDEL)*100.; FID = fopen(′INDELSummary.csv′,′a′); fprintf(FID, ′\n \n′); fprintf(FID, filename); fprintf(FID, ′\n′); fprintf(FID, num2str(INDELrate)); fid=fopen(strcat(seqsFile, ′/summary.txt′), ′wt′); fprintf(fid, ′Skipped reads %i\n not INDEL %i\n Insertions %i\n Deletions %]n INDEL percent %e\n′, [nSkips, notINDEL, NumIns, NumDels,INDELrate]); fclose(fid); save(strcat(seqsFile, ′/nSkips′), ′nSkips′); save(strcat(seqsFile, ′/notINDEL′), ′notINDEL′); save(strcat(seqsFile, ′/NumIns′), ′NumIns′); save(strcat(seqsFile, ′/NumDels′), ′NumDels′); save(strcat(seqsFile, ′/INDELrate′), ′INDELrate′); save(strcat(seqsFile, ′/dels′), ′dels′); C=dels; fid=fopen(strcat(seqsFile, ′/dels.txt′), ′wt′); fprintf(fid, ′″%s″\n′, C{:}); fclose(fid); save(strcat(seqsFile, ′/ins′), ′ins′); C=ins; fid = fopen(strcat(seqsFile, ′/ins.txt′), ′wt′); fprintf(fid, ′″%s″\n′, C{:}; fclose(fid); Supplementary Note 3.

Python script for analysis of HBG1 and HBG2 base editing and indels.

%matplotlib inline import numpy as np import scipy as sp import matplotlib as mpl import matplotlib.cm as cm import matplotlib.pyplot as plt import pandas as pd pd.set_option(′display.width′, 500) pd.set_option(′display.max columns′, 100) pd.set option(′display.notebook_repr_html′, True) import seaborn as sns sns.set_style(″whitegrid″) sns.set_context(″poster″) import requests import time from bs4 import BeautifulSoup import regex import re import os from Bio import SeqIO import Bio from Bio import motifs from Bio import pairwise2 from Bio.pairwise2 import format_alignment from Bio.Alphabet import IUPAC from sklearn import preprocessing basecall analysis with 50% Q31 cutoff on protospacer region (as defined by flanks) #includes a check for match with two HB G1 SNPs #inputs: #directory, working directory folder containing all fastq files #site, genomic site name as it appears in the fastq filenames #orientation, ′FWD′ if you want output in the same direction as the sequencing read or ′REV′ if you want reverse complement output, #flank1, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #flank2, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #width, expected bp length of basecalling window # #outputs: #′_counts.csv′, all base editing product sequences with corresponding number of occurences #′_rawsummary.csv′, summarizes base call counts for all samples #′_normalizedsummary.csv′, summarizes base call percentages for all samples def basecallhbg1(directory, site, orientation, flank1, flank2, width): indir=directory outdir=directory filenames=os.listdir(indir) for i in range(len(filenames)): seqs={} if (filenames[i][-5:]==Tastq′) and (site in filenames[i]): for record in SeqIO.parse(indir+filenames[i], ″fastq″): recordqual,[x > 31 for x in record.letter_annotations[′phred quality]] #only process reads that have more than half of basecalls >Q31 and contain two HBG1 specific SNPs at 3′ end of read if (record.seq.find(GTTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTC′) > 0) and (float(sum(recordqual))/float(len(recordqual)) > =.5): recordseq=″.join([y if x else ′N′ for (x,y) in zip(recordqual, record.seq)]) (SEQ ID NO: 86) record.seq)]) first item recordseq=″.joingy if x else ′N′ for (x,y) in zip(recordqual, #split prior to spacer window split1=recordseq.split(flank1) if len(split1)==2: #take second item in first split #split again at the sequence right after the protospacer and take split2=split1[1].split(flank2)[0] #keep only entries with exact width if (len(Split2)==width): if orientation==′FWD′: seqs[record.id],split2 elif orientation==′REV′: seqs[record.id]=Bio.Seq.reverse_complement(Split2) frame=pd.DataFrame({′Spacer′:seqs.values()}, index=seqs.keys()) Motif=motifs.create(frame.Spacer.values, alphabet=IUPAC.IUPACAmbiguousDNA()) raw=pd.DataFrame(Motif.counts, index,[str(s+1) for s in range(width)])[[′A′,′C′,′G′,′T′,′N′]].transpose() normalized=pd.DataFrame(Motif.counts, index,[str(s+1) for s in range(width)])D[[′A′,′C′,′G′,′T′]].transpose() normalized=normalized/normalized.sum(axis=0)*100. normalized=normalized.round(2) Counts=pd.DataFrame(seqs.items(), columns=[′ID′,′Window′]) Counts=Counts[[′N′, not in x for x in Counts.Window]] Counts=Counts.groupby(′Window′).count().sort_values(′ID′, ascending=False) Counts.to_csv(outdir+filenames[i].strip(′.fastq′)+′_hbg1.csv′) fd=open(directory+site+′_normalizedsummary_hbg1.csv′,′a′) fd.write(′\n′+filenames[i]+′\n′) normalized.to_csv(fd) fd.close() fd=open(directory+site+′_rawsummary_hbg1.csv′,′a′) fd.write(′\n′+filenames[i]+′\n′) raw.to_csv(fd) fd.close() return #basecall analysis with 50% Q31 cutoff on protospacer region (as defined by flanks) #includes a check for match with two HBG2 SNPs #inputs: #directory, working directory folder containing all fastq files #site, genomic site name as it appears in the fastq filenames #orientation, ′FWD′ if you want output in the same direction as the sequencing read or ′REV′ if you want reverse complement output, #flank1, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #flank2, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #width, expected bp length of basecalling window # #outputs: #′_counts.csv′, all base editing product sequences with corresponding number of occurences #′_rawsummary.csv′, summarizes base call counts for all samples #′_normalizedsummary.csv′, summarizes base call percentages for all samples def basecallhbg2(directory, site, orientation, flank1, flank2, width): indir=directory outdir=directory filenames=oslistdir(indir) for i in range(len(filenames)): seqs={} if (filenames[i][-5:]==′fastq′) and (site in filenames[i]): for record in SeqIO.parse(indir+filenames[i], ″fastq″): recordqual=[x > 31 for x in record.letter_annotations[′phred_quality′]] #only process reads that have more than half of basecalls >Q31 and contain two HBG2 specific SNPs at 3′ end of read if (record.seq.find(′ATTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTT′) > 0) and (float(sum(recordqual))/float(len(recordqual)) > =.5): recordseq=″″.join([y if x else ′N′ for (x,y) in zip(recordqual, (SEQ ID NO: 87) record.seq)]) first item #split prior to spacer window split1=recordseq.split(flank1) if len(split1)==2: #take second item in first split #split again at the sequence right after the protospacer and take split2=split1[1].split(flank2)[0] #keep only entries with exact width if (len(Split2)==width): if orientation==′FWD′: seqs[record.id]=split2 elif orientation==′REV′: seqs[record.id]=Bio.Seq.reverse_complement(Split2) frame=pd.DataFrame({′Spacer′:seqs.values()}, index=seqs.keys()) Motif=motifs.create(frame.Spacer.values, alphabet=IUPAC.IUPACAmbiguousDNA()) raw=pd.DataFrame(Motif.counts, index,[str(s+1) for s in range(width)])[[′A′,′C′,′G′,′T′,′N′]].transpose() normalized=pd.DataFrame(Motif.counts, index,[str(s+1) for s in range(width)])[[′A′,C,′G′,′T′]].transpose() normalized=normalized/normalized.sum(axis=0)*100. normalized=normalized.round(2) Counts=pd.DataFrame(seqs.items(), columns=[′ID′,′Window′]) Counts=Counts[[′N′, not in x for x in Counts.Window]] Counts=Counts.groupby(′Window′).count().sort_values(′ID′, ascending=False) Counts.to_csv(outdir+filenames[i+].strip(′.fastq′)+′_hbg2.csv′) fd=open(directory+site+′_normalizedsummary_hbg2.csv′,′a′) fd.write(′\n+filenames[i]+′\n′) normalized.to_csv(fd) fd.close() fd=open(directory+site+′_rawsummary_hbg2.csv′,′a′) fd.write(′\n′+filenames[i]+′\n′) raw.to_csv(fd) fd.close() return #indel analysis #includes a check for match with two HBG1 SNPs #inputs: #directory, working directory folder containing all fastq files #site, genomic site name as it appears in the fastq filenames #orientation, ′FWD′ if you want output in the same direction as the sequencing read or ′REV′ if you want reverse complement output, #flank1, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #flank2, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #width, expected bp length of basecalling window #ouputs: #″_Insertions_hbg1.csv″, sequences of all insertion reads #″_deletions_hbg1.csv″, sequences of all deletion reads #′indelsummary_hbg1.csv′, contains all indel stats for all fastq files def indelshbg1(directory, site, flank1, flank2, width): indir=directory outdir=directory filenames=os.listdir(indir) for i in range(len(filenames)): seqs={} if (filenames[i][-5:]==′fastq′) and (site in filenames[i]): skips=0 ins=0 insertions=[] dels=0 deletions=[] notindel=0 for record in SeqIO.parse(indir+filenames[i], ″fastq″): recordqual=[x > 31 for x in record.letter_annotations[′phred quality′]] #only process reads that have more than half of basecalls >Q31 and contain two HBG1 specific SNPs at 3′ end of read if (record.seq.find(GTTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTC′) > 0) (SEQ ID NO: 88) and (float(sum(recordqual))/float(len(recordqual))>=.5): #split prior to indel window split1=record.seq.split(flank1) if len(split1)==2: #take second item in first split #split again at the sequence right after the indel window if len(split1[1].split(flank2))==2: split2=split1[1].split(flank2)[0] #if INDEL window is +1 add to Insertions if (len(Split2)>=width+1): ins=ins+1 insertions.append(split2) #if INDEL window is -1 add to Deletions if (len(Split2)<=width-1): dels=dels+1 deletions.append(split2) if len(Split2)==width: notindel=notindel+1 else: skips=skips+1 else: skips=skips+1 else: skips=skips+1 fd=open(directory+′indelsummary_hbg1.csv′,′a′) fd.write(′\n′+filenames[i]+′\n′) fd.write(′skipped reads: ′+str(skips)+′\n′) fd.write(′insertions: ′+str(ins)+′\n′) fd.write(′deletions: ′+str(dels)+′\n′) fd.write(′notindels: ′\str(notindel)+′\′n′) fd.write(′indel rate: ′+str(float(ins+dels)/float(ins+dels+notindel)*100.)+′%′+′\n′) fd.close() pd.DataFrame(insertions).to_csv(directory+filenames[i]+′Insertions_hbg1.csv′) pd.DataFrame(deletions).to_csv(directory+filenames[i]+′Deletions_hbg1.csv′) return #indel analysis #includes a check for match with two HBG2 SNPs #inputs: #directory, working directory folder containing all fastq files #site, genomic site name as it appears in the fastq filenames #orientation, ′FWD′ if you want output in the same direction as the sequencing read or ′REV′ if you want reverse complement output, #flank1, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #flank2, sequence that is used to define the 5′ end of protospacer in the sequencing read direction, #width, expected bp length of basecalling window #ouputs: #″_Insertions_hbg2.csv″, sequences of all insertion reads #″_deletions_hbg2.csv″, sequences of all deletion reads #′indelsummary_hbg2.csv′, contains all indel stats for all fastq files def indelshbg2(directory, site, flank1, flank2, width): indir=directory outdir=directory filenames=os.listdir(indir) for i in range(len(filenames)): seqs={] if (filenames[i][-5:]==′fastq′) and (site in filenames[i]): skips=0 ins=0 insertions=[] dels=0 deletions=[] notindel=0 for record in SeqIO.parse(indir+filenames[i], ″fastq): recordqual=[x > 31 for x in record.letter_annotations[′phred_quality′]] #only process reads that have more than half of basecalls >Q31 and contain two HBG2 specific SNPs at 3′ end of read if (record.seq.find(′ATTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTT′) > 0) (SEQ ID NO: 89) and (float(sum(recordqual))/float(len(recordqual))>=.5): #split prior to indel window split1=record.seq.split(flank1) if len(split1)==2: #take second item in first split #split again at the sequence right after the indel window if len(split1[1].split(flank2))==2: split2=split1[1].split(flank2)[0] #if INDEL window is +1 add to Insertions if (len(Split2)>=width+1): ins=ins+1 insertions.append(split2) #if INDEL window is -1 add to Deletions if (len(Split2)<=width-1): dels=dels+1 deletions.append(split2) if len(Split2)==width: notindel=notindel+1 else: skips=skips+1 else: skips=skips+1 else: skips=skips+1 fd=open(directory+′indelsummary_hbg2.csv′,′a′) fd.write(′\n′+filenames[i]+′\n′) fd.write(′skipped reads: ′+str(skips)+′\n′) fd.write(′insertions: ′+str(ins)+′\n′) fd.write(′deletions: ′+str(dels)+′\n′) fd.write(′notindels: ′+str(notindel)+′\n′) fd.write(′indel rate: ′(float(ins+dels)/float(ins+dels+notindel)*100.)+′%′+′\n′) fd.close() pd.DataFrame(insertions).to_csv(directory+filenames[i]+′Insertions_hbg2.csv′) pd.DataFrame(deletions).to_csv(directory+filenames[i]+′Deletions_hbg2.csv′) return directory1=′/Users/michaelpacker/Desktop/Liu_Lab/MiSeqData/y-globin_632/′ basecallhbg1(directory1, ′632′, ′FWD′,′ATTTGCA′,′TTAATTTTTT′ (SEQ ID NO: 90), 43) basecallhbg2(directory1, ′632′, ′FWD′,′ATTTGCA′,′TTAATTTTTT′ (SEQ ID NO: 90), 43) indelshbg1(directory1, ′632′,′ATTTGCA′,′TTAATTTTTT′ (SEQ ID NO: 90),43) indelshbg2(directory1, ′632′,′ATTTGCA′,′TTAATTTTTT′ (SEQ ID NO: 90),43

EQUIVALENTS AND SCOPE, INCORPORATION BY REFERENCE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. 

What is claimed is:
 1. A method for deaminating an adenosine (A) nucleobase in a sense or antisense strand of a promoter of an HBG1 or HBG2 gene, the method comprising contacting the promoter with a base editor and a guide RNA bound to the base editor, wherein the guide RNA (gRNA) comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter of at least one of the HBG1 and HBG2 gene, wherein the base editor comprises a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain, and (ii) an adenosine deaminase, wherein the adenosine deaminase comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of a wild-type adenosine deaminase, with the exception of substitution(s) at one or more of W23, H36, N37, P48, I49, R51, N72, L84, S97, A106, D108, H123, G125, A142, S146, D147, R152, E155, I156, K157, and K161 of the amino acid sequence of SEQ ID NO: 1, or corresponding substitution(s) in another wild-type adenosine deaminase, wherein the step of contacting the promoter results in a T-A base pair in the promoter being mutated to a C-G base pair in the promoter.
 2. The method of claim 1, wherein the target nucleic acid sequence comprises: (SEQ ID NO: 838) 5′-CTTGGGGGCCCCTTCCCCACACTA-3′; (SEQ ID NO: 839) 5′-CTTGGGGGCCCCTTCCCCACACT-3′; (SEQ ID NO: 840) 5′-CTTGGGGGCCCCTTCCCCACAC-3′; (SEQ ID NO: 841) 5′-CTTGGGGGCCCCTTCCCCACA-3′; (SEQ ID NO: 842) 5′-CTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 843) 5′-CTTGGGGGCCCCTTCCCCA-3′; (SEQ ID NO: 844) 5′-CTTGGGGGCCCCTTCCCC-3′; [[or]] (SEQ ID NO: 845) 5′-CTTGGGGGCCCCTTCCC-3′; (SEQ ID NO: 297) 5′-CCACACTATCTCAATGCAAATATCTGTC-3′; (SEQ ID NO: 298) 5′-CCACACTATCTCAATGCAAATATCTGT-3′; (SEQ ID NO: 299) 5′-CCACACTATCTCAATGCAAATATCTG-3′; (SEQ ID NO: 300) 5′-CCACACTATCTCAATGCAAATATCT-3′; (SEQ ID NO: 301) 5′-CCACACTATCTCAATGCAAATATC-3′; (SEQ ID NO: 302) 5′-CCACACTATCTCAATGCAAATAT-3′; (SEQ ID NO: 303) 5′-CCGTTTCAGACAGATATTTGCAT-3′; (SEQ ID NO: 304) 5′-CCAGGGACCGTTTCAGACAGATATTTGC-3′; (SEQ ID NO: 305) 5′-CCAGGGACCGTTTCAGACAGATATTTG-3′; (SEQ ID NO: 306) 5′-CCAGGGACCGTTTCAGACAGATATTT-3′; (SEQ ID NO: 307) 5′-CCAGGGACCGTTTCAGACAGATATT-3′; (SEQ ID NO: 308) 5′-CCAGGGACCGTTTCAGACAGATAT-3′; (SEQ ID NO: 309) 5′-CCAGGGACCGTTTCAGACAGATA-3′; (SEQ ID NO: 310) 5′-ACACTATCTCAATGCAAATATCT-3′; (SEQ ID NO: 311) 5′-ACTATCTCAATGCAAATATCTGT-3′; (SEQ ID NO: 312) 5′-CCTCTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 313) 5′-CCTTGTCAAGGCTATTGGTCAAG-3′; (SEQ ID NO: 314) 5′-CCTTGCCTTGACCAATAGCCTTGACAAG-3′; (SEQ ID NO: 315) 5′-CCTTGCCTTGACCAATAGCCTTGACAA-3′; (SEQ ID NO: 316) 5′-CCTTGCCTTGACCAATAGCCTTGACA-3′; (SEQ ID NO: 317) 5′-CCTTGCCTTGACCAATAGCCTTGAC-3′; (SEQ ID NO: 318) 5′-CCTTGCCTTGACCAATAGCCTTGA-3′; (SEQ ID NO: 319) 5′-CCTTGCCTTGACCAATAGCCTTG-3′; (SEQ ID NO: 320) 5′-GCCTTGACCAATAGCCTTGACAA-3′; (SEQ ID NO: 321) 5′-CCTTGACCAATAGCCTTGACAAG-3′; (SEQ ID NO: 322) 5′-GCCTTGTCAAGGCTATTGGTCAA-3′; or (SEQ ID NO: 323) 5′-TCAAGTTTGCCTTGTCAAGGCTA-3′.


3. The method of claim 1, wherein the guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of: (SEQ ID NO: 846) 5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 847) 5′-CAUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 848) 5′-AUGUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 849) 5′-UGUGGGGAAGGGGCCCCCAAG-3′. (SEQ ID NO: 850) 5′-GUGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 851) 5′-UGGGGAAGGGGCCCCCAAG-3′; (SEQ ID NO: 852) 5′-GGGGAAGGGGCCCCCAAG-3′; or (SEQ ID NO: 853) 5′-GGGAAGGGGCCCCCAAG-3′.


4. The method of claim 1, wherein the target nucleic acid sequence comprises: (SEQ ID NO: 838) 5′-CTTGGGGGCCCCTTCCCCACACTA-3′; (SEQ ID NO: 839) 5′-CTTGGGGGCCCCTTCCCCACACT-3′; (SEQ ID NO: 840) 5′-CTTGGGGGCCCCTTCCCCACAC-3′; (SEQ ID NO: 841) 5′-CTTGGGGGCCCCTTCCCCACA-3′; (SEQ ID NO: 842) 5′-CTTGGGGGCCCCTTCCCCAC-3′; (SEQ ID NO: 843) 5′-CTTGGGGGCCCCTTCCCCA-3′; (SEQ ID NO: 844) 5′-CTTGGGGGCCCCTTCCCC-3′; or (SEQ ID NO: 845) 5′-CTTGGGGGCCCCTTCCC-3′;

and wherein deamination of the A nucleobase that is complementary to the T at position 14 of SEQ ID NO: 845 results in a T to C mutation in the target nucleic acid sequence.
 5. The method of claim 1, wherein the guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of: (SEQ ID NO: 254) 5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 255) 5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 256) 5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 257) 5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 258) 5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 259) 5′-AUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 260) 5′-AUGCAAAUAUCUGUCUGAAACGG-3′; (SEQ ID NO: 261) 5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 262) 5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 263) 5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 264) 5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 265) 5′-AUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 266) 5′-UAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 267) 5′-AGAUAUUUGCAUUGAGAUAGUGU-3′; (SEQ ID NO: 268) 5′-ACAGAUAUUUGCAUUGAGAUAGU-3′; (SEQ ID NO: 269) 5′-GUGGGGAAGGGGCCCCCAAGAGG-3′; (SEQ ID NO: 270) 5′-CUUGACCAAUAGCCUUGACAAGG-3′; (SEQ ID NO: 271) 5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 272) 5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 273) 5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 274) 5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 275) 5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 276) 5′-CAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 277) 5′-UUGUCAAGGCUAUUGGUCAAGGC-3′; (SEQ ID NO: 278) 5′-CUUGUCAAGGCUAUUGGUCAAGG-3′; (SEQ ID NO: 279) 5′-UUGACCAAUAGCCUUGACAAGGC-3′; (SEQ ID NO: 280) 5′-UAGCCUUGACAAGGCAAACUUGA-3′.


6. The method of claim 5, wherein the target nucleic acid sequence comprises (SEQ ID NO: 297) 5′-CCACACTATCTCAATGCAAATATCTGTC-3′; (SEQ ID NO: 298) 5′-CCACACTATCTCAATGCAAATATCTGT-3′; (SEQ ID NO: 299) 5′-CCACACTATCTCAATGCAAATATCTG-3′; (SEQ ID NO: 300) 5′-CCACACTATCTCAATGCAAATATCT-3′; (SEQ ID NO: 301) 5′-CCACACTATCTCAATGCAAATATC-3′; or (SEQ ID NO: 302) 5′-CCACACTATCTCAATGCAAATAT-3′;

and wherein deamination of the A nucleobase that is complementary to the T at position 21 of SEQ ID NO: 302 results in a T to C mutation in the target nucleic acid sequence.
 7. The method of claim 5 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein: (a) the second target nucleic acid sequence comprises SEQ ID NO: 845; (b) the second guide sequence comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 853; or (c) both (a) and (b).
 8. The method of claim 1, wherein the guide sequence of the gRNA comprises at least 15 contiguous nucleotides of the sequence (SEQ ID NO: 270) 5′-CUUGACCAAUAGCCUUGACAAGG-3′.


9. The method of claim 8, wherein the target nucleic acid sequence comprises 5′-CCTTGTCAAGGCTATTGGTCAAG-3′ (SEQ ID NO: 313); wherein deamination of an A nucleobase that is complementary to the T at any one of positions 15, 16, or 19 of SEQ ID NO: 313 results in a T to C mutation in the target nucleic acid sequence.
 10. The method of claim 8 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein: (a) the second target nucleic acid sequence comprises SEQ ID NO: 845, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 309, SEQ ID NO: 311, or SEQ ID NO: 312; (b) the second guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 853, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 268, and SEQ ID NO: 269; or (c) both (a) and (b).
 11. The method of claim 1 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein: (a) the second target nucleic acid sequence comprises SEQ ID NO: 845, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 319, SEQ ID NO: 320, or SEQ ID NO: 321; (b) the second guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 853, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278; or (c) both (a) and (b).
 12. The method of claim 1, wherein the step of contacting the promoter leads to an increase in transcription of the HBG1 gene or the HBG2 gene.
 13. The method of claim 1, wherein the method is performed in a cell of a subject who has a disease or disorder of the blood.
 14. The method of claim 13, wherein the disease or disorder is caused by a mutation in a gene, or a promoter of a gene, selected from the group consisting of CYGB, HBA1, HBA2, HBB, HBD, HBE1, HBG1, HBG2, HBM, HBQ1, HBZ, and MB.
 15. The method of claim 1, wherein the adenosine deaminase comprises each of the W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N substitutions in SEQ ID NO: 1, or the corresponding substitutions in another wild-type adenosine deaminase.
 16. The method of claim 1, wherein the guide sequence of the guide RNA comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of: (SEQ ID NO: 254) 5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 255) 5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 256) 5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 257) 5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 258) 5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 259) 5′-AUAUUUGCAUUGAGAUAGUGUGG-3′; (SEQ ID NO: 260) 5′-AUGCAAAUAUCUGUCUGAAACGG-3′; (SEQ ID NO: 261) 5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 262) 5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 263) 5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 264) 5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 265) 5′-AUAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 266) 5′-UAUCUGUCUGAAACGGUCCCUGG-3′; (SEQ ID NO: 267) 5′-AGAUAUUUGCAUUGAGAUAGUGU-3′; (SEQ ID NO: 268) 5′-ACAGAUAUUUGCAUUGAGAUAGU-3′; (SEQ ID NO: 269) 5′-GUGGGGAAGGGGCCCCCAAGAGG-3′; (SEQ ID NO: 270) 5′-CUUGACCAAUAGCCUUGACAAGG-3′; (SEQ ID NO: 271) 5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 272) 5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′ ; (SEQ ID NO: 273) 5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 274) 5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 275) 5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 276) 5′-CAAGGCUAUUGGUCAAGGCAAGG-3′; (SEQ ID NO: 277) 5′-UUGUCAAGGCUAUUGGUCAAGGC-3′; (SEQ ID NO: 278) 5′-CUUGUCAAGGCUAUUGGUCAAGG-3′; (SEQ ID NO: 279) 5′-UUGACCAAUAGCCUUGACAAGGC-3′; or (SEQ ID NO: 280) 5′-UAGCCUUGACAAGGCAAACUUGA-3′.


17. The method of claim 1, wherein the method causes less than 1% indel formation.
 18. The method of claim 1, wherein the efficiency of deaminating an A nucleobase is at least 30%.
 19. The method of claim 1, wherein the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to SEQ ID NO:
 270. 20. The method of claim 19, wherein the nucleobase changes comprise a deletion of one or more nucleobases of SEQ ID NO:
 270. 21. The method of claim 20, wherein the gRNA comprises the nucleic acid sequence 5′-CUUGACCAAUAGCCUUGACA-3′ (corresponding to bases 1-20 of SEQ ID NO: 270).
 22. The method of claim 20, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 23. The method of claim 1, wherein the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to SEQ ID NO:
 260. 24. The method of claim 19, wherein the nucleobase changes comprise a deletion of one or more nucleobases of SEQ ID NO:
 260. 25. The method of claim 24, wherein the gRNA comprises the nucleic acid sequence 5′-AUGCAAAUAUCUGUCUGAAA-3′ (corresponding to bases 1-20 of SEQ ID NO: 260).
 26. The method of claim 24, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 27. The method of claim 2, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 28. The method of claim 4, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 29. The method of claim 6, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 30. The method of claim 7, wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 31. The method of claim 9, wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 32. The method of claim 10, wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 33. The method of claim 11, wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
 34. The method of claim 1, wherein the adenosine deaminase comprises one or more of W23L, W23R, H36L, N37S, P48S, P48A, I49V, R51L, N72D, L84F, S97C, A106V, D108N, H123Y, G125A, A142N, S146C, D147Y, R152P, E155V, I156F, K157N, and/or K161T substitutions in SEQ ID NO: 1, or one or more corresponding substitutions in another wild-type adenosine deaminase.
 35. The method of claim 1, wherein the adenosine deaminase comprises one or more of W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and/or K157N substitutions in SEQ ID NO: 1, or one or more corresponding substitutions in another wild-type adenosine deaminase.
 36. The method of claim 1, wherein the adenosine deaminase comprises A106V and D108N substitutions in SEQ ID NO: 1, or corresponding substitutions in another wild-type adenosine deaminase.
 37. The method of claim 1, wherein the adenosine deaminase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 65-83, 420-437, 672-684, and 802-805.
 38. The method of claim 1, wherein the adenosine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 65-83, 420-437, 672-684, and 802-805.
 39. The method of claim 1, wherein the napDNAbp domain comprises the amino acid sequence of any one of SEQ ID NOs: 34-36 or 417-419. 